Beta-agonist compounds comprising nitric oxide donor groups and reactive oxygen species scavenger groups and their use in the treatment of respiratory disordersB

ABSTRACT

The present invention relates to multifunctional β-agonist compounds comprising a reactive oxygen species scavenger group and a nitric oxide donor, and to methods of treating chronic obstructive airway diseases, such as asthma and chronic bronchitis.

FIELD OF THE INVENTION

The present invention relates to multifunctional β-agonist compoundsthat are capable of acting both as nitric oxide donors and as scavengersof reactive oxygen species and which are useful in the treatment ofrespiratory disorders. The invention further relates to methods of usingsuch compounds in the treatment of respiratory disorders such as asthma.

BACKGROUND OF THE INVENTION

NO (nitric oxide) is formed from the amino acid L-arginine by severalforms of NO synthases, and plays a role in a number of physiologicalfunctions, including the relaxation of airway smooth muscle. NO formedin endothelial cells in response to chemical agonists and to physicalstimuli plays a key role in regulation of vascular tone, plateletaggregation and adhesion, as well as modulating smooth muscleproliferation (Haj-Yehia et al. (2000) Drug. Development Res.50:528-536). NO overproduction has also been associated with numerousdisease states (WO 99/66918). NO levels have been shown to be increasedin the asthmatic airways (Kaminsky et al. (1999) J. Allergy Clin.Immunol 104(4)I:747-754). The role of NO in the respiratory system hasbeen studied (Tamaoki et al. (1995) Am. J. Physiol.268(6)I:C1342-C1346). NO has also been used in the treatment ofasthmatics, though such treatments demonstrated a great deal of inter-and intra-individual variability (WO 01/32202).

Publications disclosing nitric oxide donor compounds or compounds whichpromote the synthesis of nitric oxide include WO 98/42661, WO 99/37616,WO 00/31060, WO 97/34871, WO 00/35434, WO 99/62509, WO 97/25984, WO00/67754, WO 9961018, WO 99/61430, WO 97/31654, WO 96/32946, WO00/53191, U.S. Pat. Nos. 6,248,895 and 6,232,331 and Wolf et al. (1998)J. Neurosurg. 89:279-288. Publications disclosing nitric oxide scavengercompounds include WO 98/55453.

The endothelium, in addition to producing NO, also produces superoxide(SO) anion and other reactive oxygen species (ROS) under physiologicalconditions. Despite SO being a reducing agent that is itself incapableof initiating oxidative reactions, SO is considered the most importantsource of oxidative stress. Compounds for the removal of SO aredescribed in the art, including WO 96/39409 and U.K. Pat. App. No.2349385A.

Many disease states, including diabetes mellitus and variouscardiovascular diseases, are associated with oxidative stress andendothelial dysfunction. Nitroglycerin (GTN) has been used for thetreatment of various types of myocardial ischemia. Because of itspathogenic nature (chronicity with acute exacerbation), prophylactic andacute treatments are necessary to prevent complications with potentiallyfatal outcomes (>25% death for acute MI). However, the phenomenon oftolerance to the anti-anginal effects of GTN and to all other existingorganic nitrates is of a special clinical significance. In particular,early development of tolerance to the drug is by far the most seriousdrawback of nitrate therapy.

A number of respiratory disorders have been recognized. Many of whichhave overlapping and interacting etiologies. The majority of thesedisorders are characterized by acute pulmonary vasoconstriction orbronchoconstriction. Inflammation and edema are also often associatedwith respiratory disorders such as asthma, respiratory distress syndrome(child or adult), bronchitis, pneumonia and others.

Various compounds and treatments for respiratory disorders are disclosedin the art, for example, in U.S. Pat. Nos. 6,299,863, 6,124,319,6,297,762, 6,254,882, 6,083,993, 5,824,669, 5,821,259, RE 37,116E, WO97/34871, WO 01/32202, WO 99/40787, WO 95/30641 and Australian PatentNo. 733202.

At the therapeutic level, β₂-agonists are the first drugs that are usedin the acute treatment of asthma (Roberts et al., Lung (1990) 168:Suppl.105-110, and Rees, BMJ (1991) 302:1166-7). It is more controversialwhether they should be used for chronic maintenance therapy (Haahtela etal. NEJM (1991) 325:388-92 and Burrows and Lebowitz, NEJM (1992)326:560-1). Several effective β₂-agonists are currently available.However, patients may respond better to one drug over another so it isreasonable to switch drugs if a patient is not responding (Thompson etal., Clin. Pharm. 1985; 4:383-8). The inhaled route of administration isthe preferred route and a metered dose inhaler is the preferred way ofadministering the drug by inhalation. Methods of making aerosolformulations of β₃-agonists are known in the art, as described forexample in U.S. Pat. No. 6,238,647.

Most β₂-agonists cause somewhat similar adverse effects. These adverseeffects include but are not limited to cardiovascular effects such aspalpitations, increased heart rate, and tachycardia; central nervoussystem symptoms such as nervousness, dizziness, headache and drowsiness;respiratory side effects such as dyspnea, wheezing, drying or irritationof the oropharynx, coughing, chest pain and chest discomfort; handtremors, muscle tremors, and immediate hypersensitivity reactions suchas urticaria, angioedema, rash and even bronchospasms. In addition, someβ₂-agonists can cause angina, vertigo, central stimulation and insomnia,airway hyperreactivity (hypersensitivity), nausea, diarrhea, dry mouthand vomiting (see also Boushey, “Basic & Clinical Pharmacology” 7th Ed.,pp. 118-151 and 325-342, Katzung, Ed. 1998, Appleton & Lange, Stamford,Conn.). β₂-agonists may sometimes cause systemic adverse effects such asweakness, fatigue, flushed feeling, sweating, unusual taste, hoarseness,muscle cramps and backaches.

Furthermore, patients have a tendency to develop a tolerance to thebronchodilating effect of β-agonists. This is related todesensitization, which is one of the most clinically significantphenomena involving the β-adrenergic receptor. It has been observed thatpatients in prolonged β-agonist therapy have a tendency to increase thedosage their medication. This occurs because after prolongedadministration, the β-receptor appears to become desensitized to theagonist, thus requiring larger doses of the compound to effect anequivalent physiological response. Often an increase in dosage leads toan increase in the kind, number or severity of adverse effects (see e.g.Paterson et al: American Review of Respiratory Disease (1979)120:844-1187); Lancet (1990) 336:1411-1412; Spitzer et al. New EnglandJ. Med. (1992).

There is a need for improved drugs for the treatment of respiratorydisorders such as asthma.

SUMMARY OF THE INVENTION

The present invention relates to multifunctional β-agonist compoundsthat are capable of acting both as nitric oxide donors and as scavengersof reactive oxygen species and which are useful in the treatment ofrespiratory disorders. The invention further relates to methods of usingsuch compounds in the treatment of respiratory disorders such as asthma.

In one embodiment a multifunctional β-agonist compound is providedcomprising a β-agonist component, at least one antioxidant, such as areactive oxygen species (ROS) scavenger component, and at least onenitric oxide donor component.

In one embodiment a multifunctional β-agonist compound is providedcomprising a β-agonist for the treatment of respiratory disorders linkedto at least one reactive oxygen species (ROS) scavenger and at least onenitric oxide donor.

In certain embodiments, the β-agonist provided is based on modifiedsalbutamol, salmetrol, fometeral, or terbutalin.

In other embodiments of the compounds as described herein the compoundcomprises at least two nitric oxide donors.

In other embodiments of the compounds as described herein, the nitricoxide donor is independently selected from the group consisting of —ONO,—ONO₂, —SNO and —NONOate.

In still other embodiments, the antioxidant, such as an ROS scavenger,is a substituted nitroxide free radical; alkenyl group; aryl group;substituted aryl group, where the aryl group is substituted with, forexample, —OH, —NH₂, —NHCHO or a NO donor group; or a group that is, oris capable of being converted in vivo into, a sulfhydryl in oxidized orreduced form.

Compounds in one embodiment are provided of Formula 1:

-   -   wherein R¹ is selected from the group consisting of —OH, —ONO,        —ONO₂, —SNO, and —NONOate;    -   R² is optionally substituted alkyl; or a group comprising an ROS        scavenger and optionally an NO-donor;    -   R³ and R⁴ are independently selected from the group consisting        of —OH, —CH₂OH, —NH₂, and —NHCHO; or R³ and R⁴ together form a        substituted 5 to 7-membered saturated heterocycle having 1 or 2        heteroatoms independently selected from nitrogen, and oxygen,        and sulfur, e.g. a substituted pyrrolidine, substituted        oxazolidine or substituted piperidine; or R³ and R⁴ together        form amino or hydroxy protecting groups selected from N-formyl,        acetal, and ketal; and    -   R⁵ is selected from the group consisting of —H, —OH, —CH₂OH,        —NH₂, —NHCHO, straight or branched chain C₁-C₃ alkyl and        straight or branched chain C₁-C₃ alkoxy;    -   whereas said RO scavenger moieties are optionally substituted        with one or more C₁-C₁₅ alkyl groups, C₁-C₁₅ alkoxy groups,        phenyl, —NH₂, —NHCHO, —OH, —CH₂OH, and groups capable of        donating NO in a charged or neutral form; and    -   whereas any of said alkyl groups is optionally substituted with        one or more functional groups selected from hydroxyl, bromo,        fluoro, chloro, iodo, mercapto or thio, cyano, alkylthio, aryl,        carboxyl, carbalkoyl, alkenyl, nitro, amino, alkoxyl, amido;        wherein at least one of R¹, R², R³ or R⁴ comprises at least one        ROS scavenger; and        wherein at least one of R¹, R², R³ or R⁴ comprises at least one        NO donor.

In certain embodiments of the compound described by the above formula,R¹ is —ONO₂ or —SNO.

In other embodiments of the compound as described by the above formulaR² is substituted with —ONO₂ or —SNO.

In other embodiments of the compounds of Formula 1, R³ and R⁴ togetherform a substituted pyrrolidine, substituted oxazolidine or substitutedpiperidine N-oxide free radical.

Other embodiments include compounds having the formula

wherein R¹ is selected from the group consisting of —OH, —ONO, —ONO₂,—SNO, and —NONOate;

R² is optionally substituted alkyl; or a group comprising an antioxidantand optionally an NO-donor;

wherein R² is optionally substituted with —ONO, —ONO₂, or —SNO; and

R⁵ is selected from the group consisting of —H, C₁-C₃ alkyl, C₁-C₃alkoxy, —OH, —CH₂OH, —NH₂, and —NHCHO.

In particular embodiments of the compound as described by the aboveformula, R¹ is —ONO₂ or —SNO.

In other embodiments of the compound as described by the above formula,R² comprises an aryl group.

In still other embodiments of the compounds as described by the aboveformulas, R² is selected from the group consisting of t-butyl,

In one embodiment, the following compounds, and methods for their use inthe treatment of respiratory disorders are provided:

and salts thereof.

Included in the scope of the present invention are compositionscomprising the compounds as described herein and a pharmaceuticallyacceptable excipient.

Also included herein are methods of treating a respiratory condition inan individual in need thereof comprising administering a therapeuticallyeffective amount of a compound as described herein to said individual.

The present invention provides the compounds of the present inventionfor use in therapy.

The present invention further provides use of the compounds of thepresent invention in the manufacture of a medicament for the treatmentof respiratory disorders.

In certain embodiments of the methods described herein, a compound orcomposition as described herein is administered is orally. The compoundor composition may be administered by inhalation.

In some embodiments of the methods described herein, the respiratorycondition is asthma, chronic obstructive pulmonary disease, bronchialhyperreactivity, adult respiratory distress syndrome, emphysema,bronchopulmonary dyspasia, or interstitial pulmonary fibrosis.

Certain embodiments include an inhalation device comprising a compoundas described herein. In still other embodiments, a kit is provided forthe treatment of respiratory disorders including a multifunctionalβ-agonist compound, an inhalation device, and optionally, appropriatepackaging, labeling and/or instructions for use, wherein the inhalationdevice is optionally a metered dose inhaler.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relaxation response (relax. %) of salbutamol (sal.,open squares), compound 2 (comp.2, filled circles) and itsnon-nitrosolated precursor, compound 2a (comp. 2a, open triangles)versus log of concentration (log (conc.)).

FIG. 2 shows the cGMP response of tracheal rings to a control (cont.),salbutamol, compound 2a (non-nitrosolated precursor of compound 2),compound 2, and compound 2 in the presence of methylene blue (MB).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Provided are multifunctional β-agonist compounds for the treatment ofrespiratory disorders. The multifunctional β-agonist compound includesin one embodiment: a β-agonist component; an antioxidant, such as areactive oxygen species scavenger; and a nitric oxide donor. Thus, inone embodiment, a β-agonist is provided in modified form and includes areactive oxygen species (ROS) scavenger group and a nitric oxide donorgroup capable of releasing NO in a charged or neutral form. Theβ-agonist may be linked to at least one reactive oxygen species (ROS)scavenger and at least one nitric oxide donor. Exemplary β-agonistsinclude salbutamol, salmetrol, fometeral, and terbutalin. The nitricoxide donors include —ONO, —ONO₂, —SNO and —NONOate. The antioxidantgroup, such as a ROS scavenger is, for example, a substituted nitroxidefree radical; alkenyl group; aryl group; substituted aryl group, wherethe aryl group is substituted with, for example, —OH, —NH₂, —NHCHO or aNO donor group; or a group that is, or is capable of being converted invivo into, a sulfhydryl in oxidized or reduced form.

The multifunctional β-agonist compounds, and compositions comprising themultifunctional β-agonist compounds, may be used in methods of treatingrespiratory disorders including asthma, bronchitis, emphysema,bronchospasms, pneumonia, bronchial hyper-reactivity, respiratorydistress syndrome and other ailments in patients with obstructive airwayor allergic disorders. The multifunctional β-agonist compounds,compositions comprising the multifunctional β-agonist compounds andmethods described herein are also directed to avoiding adverse effects,development of tolerance (e.g. desensitization) or hypersensitivity onrepeated administration. The multifunctional β-agonist compounds andcompositions comprising the multifunctional β-agonist compounds asdescribed herein may also be used in the manufacture of medicaments forthe treatment of respiratory disorders.

The multifunctional β-agonist compounds, and compositions comprising themultifunctional β-agonist compounds, described herein not only provide asource of nitric oxide, which acts in the regulation of airway smoothmuscle, but in acting as an antioxidant scavenger of superoxide anionand other reactive oxygen species give rise to both a direct benefitderived from removal of injurious superoxide anion and other reactiveoxygen species and a benefit in protecting both ambient and endogenousand liberated exogenous NO from inactivation by superoxide anion andother reactive oxygen species.

As used herein, the term “multifunctional β-agonist compound” refers toa compound containing a β-agonist component, and additionally at leastone NO donor component and at least one antioxidant component, such asan ROS scavenger component. The components may be linked, for exampledirectly, indirectly and/or via a sharing of atoms, as described herein.The use of the term “multifunctional β-agonist compound” is not intendedto necessarily require that the compound was formed by chemicalmodification of a β-agonist, since the synthesis would not necessarilyinvolve a starting material that was a β-agonist that is furthermodified, and other routes of synthesis are contemplated. Rather, a“multifunctional β-agonist compound” is meant to be a molecule that notonly includes a β-agonist component with β-agonist activity, but alsothe additional functionality of the NO donor and antioxidant (such asROS scavenger) components. Thus, in one embodiment, multifunctionalβ-agonist compounds are provided that are β-agonists in a modified formwherein they include an NO donor group and a ROS scavenger group. It isto be understood that the compounds of the invention whether preparedfrom a β-agonist as a starting material or any other starting materialretain their β-agonistic activity.

NO Donors

Groups that can act as nitric oxide donors are capable of acting as asource of nitric oxide (NO). The nitric oxide donor is, for example, an—ONO₂—ONO, —SNO or —NONOate group. In particular embodiments the NOdonor is —ONO₂ or —SNO. The NO donor, for example, donates, transfers,or releases nitric oxide in either a neutral or a charged form. Thenitric oxide donor may comprise any group capable of acting as a sourceof nitric oxide (NO) in a charged or uncharged form, includingnitrosonium (NO⁺), nitroxyl (NO⁻) or nitric oxide (NO.).

Reactive Oxygen Species Scavengers

The multifunctional β-agonist compound may include an antioxidant groupthat can act as an antioxidant, preferably without reacting with nitricoxide, as well as the NO donor. The antioxidant group can be a reactiveoxygen species (ROS) scavenger. As used herein, the term “reactiveoxygen species (ROS) scavenger group” refers to a group capable ofacting as a scavenger of, or reacting with, superoxide (O₂ ⁻) or otherreactive oxygen species (ROS) including hydroxyl radicals,peroxynitrite, hypochlorous acid and hydrogen peroxide. An antioxidantthat preferentially scavenges, or reacts with, superoxide is termed a“superoxide dismutase mimic” (“SOD-mimic”) or “superoxide dismutasemimetic” (“SOD-mimetic). The reactive oxygen species superoxide (O₂ ⁻),hydroxyl radicals, peroxynitrite, hypochlorous acid and hydrogenperoxide are considered biologically undesirable, while nitric oxide, asdescribed above, may be biologically beneficial. Thus, the antioxidantor ROS scavenger group preferably does not react with, or scavenge,nitric oxide.

The multifunctional β-agonist compounds described herein may include oneor more antioxidant or ROS scavenger groups. In some embodiments, thereactive oxygen species scavenger is a nitroxide free radical (NO.)group. In certain embodiments the compounds as described herein maycomprise more than one ROS scavenger, for example at least one, at leasttwo, at least three or at least four ROS scavenger groups.

As used herein, the ROS scavenger itself is not intended to be a groupcapable of donating nitric oxide (NO). Further, the ROS scavenger isprovided in addition to the β-agonist component of the multifunctionalβ-agonist compound.

The antioxidant group, such as an ROS scavenger group, may be forexample an alkenyl group; aryl group; substituted aryl group, where thearyl group is substituted with, for example, —OH, —NH₂, —NHCHO or a NOdonor group; sulfhydryl or dithiol in oxidized or reduced form; or agroup that is, or is capable of being converted in vivo into, asulfhydryl in its oxidized or reduced form.

As used herein, the term “alkyl” includes branched or unbranchedhydrocarbon chains, for example, including about 1 to about 18 carbons,or 1-15, or 1-10 or 1-5 carbons, such as methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, iso-butyl, tert.-butyl, octa-decyl and2-methylpentyl. Alkyl may also include cyclic alkyl groups, for example,including about 5-8 carbons, such as cyclopentyl, cyclohexyl,cycloheptyl, or cycloctyl. Alkyl can be optionally substituted with oneor more functional groups such as hydroxyl, bromo, fluoro, chloro, iodo,mercapto or thio, cyano, alkylthio, aryl, carboxyl, carbalkoyl, alkenyl,nitro, amino, alkoxyl, amido, an NO donor group, and the like in theform of substituted alkyl. A cyclic alkyl group may be substituted witha straight or branched chain alkyl group.

The term “aryl” includes a chain of carbon atoms which form at least onearomatic ring having for example between about 6-14 carbon atoms, suchas phenyl, naphthyl, anthracenyl and azulenyl.

The aryl optionally may be substituted with one or more functionalgroups such as hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio,cyano, cyanoamido, alkylthio, heterocycle, aryl, heteroaryl, carboxyl,carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, NO donorgroups, and the like.

The term “heteroaryl” includes a ring system including one or morearomatic rings and containing one or more heteroatoms, N, O, or S, inthe aromatic ring. Heteroaryl groups can be unsubstituted or may besubstituted for example as described for alkyl and aryl groups. Examplesof heteroaryl groups include, but are not limited to, pyridinyl,pyrazinyl, pyrimidinyl, benzothialozyl, pyrazolyl, benzoxazolyl,imidazolyl, pyrrolyl, thiadiazolyl, oxazolyl, isoxazolyl, pyridazinyl,triazolyl, thiazolyl, isothiazolyl, thiophenyl, furanyl, and quinolinyl.

In particular embodiments, the ROS scavenger may be a nitroxide freeradical, wherein optionally the N is within a 3-, 4-, 5-, 6- or7-membered ring, wherein the ring may be optionally substituted with,for example, straight or branched chain C₁-C₃ alkyl groups, alkoxygroups and groups capable of donating NO.

The nitroxide free radical is preferably substituted. In particularembodiments the nitroxide free radical is fully substituted at positionsalpha to the nitroxide free radical, and may optionally be substitutedat other positions on the ring. Exemplary substituents for the alphapositions include methyl or ethyl. Exemplary substituents for other ringpositions include NO donor groups.

In certain other embodiments the substituted nitroxide free radical mayalso be substituted within the ring with an additional heteroatom, forexample, —O— or —S—. (see structures Ia and Ib, below). Exemplarynitroxide free radicals include substituted pyrrolidinyloxy freeradicals (e.g. PROXYL), substituted piperidinyloxy free radicals (e.g.TEMPO), substituted oxazolidinyloxy free radicals (e.g. DOXYL),oxazinyloxy free radicals, substituted thiazolidinyloxy free radicalsand substituted thiazinyloxy free radicals.

In certain embodiments, the ROS scavenger(s) may be independentlyselected from the group consisting of substituted piperidinyloxy freeradical, substituted 3-pyrrolidin-1-yloxy free radical, substitutedoxazolidinyloxy free radical (e.g. DOXYL), and an optionally substitutedlipoic acid moiety.

Examples of nitroxide free radical moieties which may be incorporatedinto the multifunctional β-agonist compounds include a2,2,6,6-tetramethylpiperidinyloxy free radical (TEMPO) moiety (Ia,below, where X=C), a 2,2,5,5-tetramethyl-3-pyrrolidin-1-yloxy freeradical (PROXYL) moiety (Ib, below, where X=C);4,4-dimethyl-3-oxazolidinyloxy (DOXYL) free radical moiety, and a2,2,4,4-tetramethyl-3-oxazolidinyloxy free radical moiety (Ib, below,where X=O). In structures Ia-f below, X is for example —S—, —C— or —O—.The nitroxide free radical moiety may be linked to the β-agonist moietyfor example, directly, indirectly, via a linker (e.g. through an alkylsubstituent group, see, for example Ic and Id), and/or via sharing ofatoms, for example as shown in Formulas 1a-1e or structures 1e and 1fbelow. The linkage may be to various carbon atoms on the ring, includingthose shown in structures Ic-If below.

In other embodiments the ROS scavenger comprises a lipoic acid moiety ormay be derived from the lipoic acid moiety. The lipoic acid moiety maybe optionally substituted and is shown below:

The lipoic acid moiety may be independently substituted by one or moregroups such as straight or branched chain C₁-C₁₅ alkyl groups, C₁-C₁₅alkoxy groups, hydroxy groups, amino groups (NH₂), —NHCHO groups, —CH₂OHgroups, and groups capable of donating NO in a charged or neutral form.

In other embodiments, the ROS scavenger may be a pantothenic acidSH-containing derived moiety as shown below, in either an oxidized orreduced form:

wherein, m is for example, 1-6, and R⁸ and R⁹ are for example eachindependently C₁-C₃ alkyl or H.

In other embodiments, the lipoic acid moiety may be modified by varyingthe length of the aliphatic chain connecting the heterocyclic ring tothe β-agonist component of the multifunctional β-agonist compound. Thechain may be for example (CH₂), wherein n is an integer from 1-15. Incertain embodiments n is 2-12, 5-12, or 8-12. In particular embodiments,n is 3 or 12 as shown below.

In certain embodiments the ROS scavenger, including those describedabove, may be independently substituted with one or more alkyl groupssuch as C₁-C₁₅ alkyl groups, alkoxy such as C₁-C₁₅ alkoxy groups,hydroxy groups, amino groups including NH₂, —NHCHO groups, —CH₂OHgroups, and groups capable of donating NO in a charged or neutral form.

In other embodiments, the antioxidant may comprise or be one or moreoptionally substituted aryl groups or heteroaryl groups. In certainembodiments the aryl group is phenyl. The aryl groups may be optionallyindependently substituted by one or more groups including straight orbranched chain C₁-C₁₅ alkyl groups, C₁-C₁₅ alkoxy groups, hydroxygroups, amino groups (such as NH₂), —NHCHO groups, —CH₂OH groups, andgroups capable of donating NO in a charged or neutral form.

In particular embodiments, the ROS scavenger group(s) comprises, one ormore PROXYL moieties, one or more TEMPO moieties, one or more DOXYLmoieties, one or more 2,2,4,4-tetramethyl-3-oxazolidinyloxy free radicalmoieties and/or one or more optionally substituted lipoic acid moieties.In particular embodiments the groups comprising N-oxide free radicalmoieties are independently substituted by one or more C₁-C₃ alkylgroups, for example methyl, ethyl or butyl, or one or more C₁-C₃ alkoxygroups.

The multifunctional β-agonist compounds may be modified to include oneor more of the same or different antioxidant and/or ROS scavenger group.

β-Agonists

The β-agonist component of any of a variety of β-agonist compounds forthe treatment of respiratory disorders can be present in themultifunctional β-agonist compounds. In one embodiment, a knownβ-agonist is provided in multifunctional form which further includes atleast one NO donor and at least one ROS scavenger group. The β-agonistcompound or component is one that is capable of exertingbronchorelaxation through stimulation of β-adrenoreceptors.

Exemplary β-agonists include compounds used in the treatment ofrespiratory disorders that are selective for the β₂ adrenergicreceptors, such as terbutaline, albuterol, salbutamol, fenoterol,isoetharine, metaproterenol and, the so-called “long acting” selectiveβ₂ sympathomimetic bronchodilator compounds formoterol, bambuterol andsalmeterol. Another β-agonist is isoproterenol. A further class ofβ-agonists include the bronchorelaxants which are ephedrine derivatives,(e.g. albuterol, bambuterol, bitolterol, carbuterol, clenbuterol,clorprenaline, dioxethedrine, ephedfine, epinephrine, eprozinol,etafedrine, ethylnorepinephrine, fenoterol, formoterol, hexoprenaline,isoetharine, isoproterenol, mabuterol, metaproterenol,N-methylephedrine, pirbuterol, procaterol, protokylol, reproterol,rimiterol, salmeterol, soterenol, terbutaline, and tulobuterol).

Numerous β₂ sympathomimetic bronchorelaxant drugs are commerciallyavailable and clinically used, generally in pharmaceutically acceptablesalt form, e.g., as the sulphate, hydrobromide, hydrochloride, fumarateor methanesulfonate or, where appropriate, one or other of the hydrateforms thereof. Beta agonists are useful as bronchodilator agents; theystimulate β₂ adrenergic receptors, increase intracellular cAMP, andinhibit the release of mast cell mediators, which can causebronchoconstriction (Boushey, “Basic & Clinical Pharmacology” 7th Ed.,pp. 118-151 and 325-342, Katzung, Ed. 1998, Appleton & Lange, Stamford,Conn.). Additional β-agonists used in the treatment of respiratorydisorders are known in the art and may be used as described herein, forexample, those described in “Sympathomimetic Enantiomers in theTreatment of Asthma” Ed. J. F. Costello (1997), Parthenon PublishingGroup, Inc., London and “New Drugs for Asthma, Allergy and COPD”, Ed. T.Hansel and P. J. Barnes (2001) Karger, London.

Albuterol acts selectively on β₂-adrenergic receptors to relax smoothmuscle tissue, for example, in the bronchial system. Albuterol, alsoknown as(alpha1-((tert.-butylamino)methyl)-4-hydroxy-m-xylene-alpha,alpha-diol)is commonly used to treat bronchial spasms associated with asthma and isthe active component in well-known commercial bronchodilators such asProventil™ and Ventolin™. Other formulations of albuterol includeAerolin™, salbulin, ventodisks, salbutamol, asmol, respax, respolin.

In certain examples the β-agonist compound or component elicits abronchodilator effect, such as relief from the symptoms associated withobstructive airway diseases, which include but are not limited torespiratory distress, wheezing, coughing, shortness of breath, tightnessor pressure in the chest and the like.

The problem of desensitization is particularly significant in thetreatment of diseases involving bronchospasms, such as asthma, uponself-administration either orally or by aerosol of β-adrenergicagonists. Such β-agonists may be provided in a multifunctional form asdisclosed herein.

Multifunctional β-Agonist Compounds

The multifunctional β-agonist compound may include an β-agonistcomponent, and additionally, at least one NO-donor, and at least oneantioxidant such as a reactive oxygen species (ROS) scavenger. Themultifunctional β-agonist compound may include a β-agonist componentlinked to at least one NO-donor and at least one antioxidant. The term“linked” as used herein is intended to include direct and indirectlinkages and shared atoms between any of the NO donor group,antioxidant, such as ROS scavenger group, and β-agonist component. Thecomponents may be linked in any order, for example, the ROS scavengermay be linked to both the NO donor and the β-agonist component, or theROS scavenger may be linked only to the β-agonist component while theβ-agonist component is also linked to the NO-donor (e.g. according toFormula 1).

Also included within the scope of the invention are salts of thecompounds disclosed herein and stereoisomers thereof. The compounds ofthe present invention contain one or more asymmetric atoms and may existin diastereomeric, racemic and optically active forms. All suchcompounds and compositions comprising these compounds are contemplatedto be within the scope of this invention. Therefore, where a compound ischiral, the separate enantiomers, substantially free of the other, areincluded within the scope of the invention. Thus, one enantiomer may bein, for example, 95% or more purity. Further included are all mixturesof enantiomers or diastereomers.

Optically active forms of the compounds can be prepared using any methodknown in the art, including by resolution of the racemic form byrecrystallization techniques, by chiral synthesis, extraction withchiral solvents, or by chromatographic separation using a chiralstationary phase. Examples of methods to obtain optically activematerials include transport across chiral membranes, a technique wherebya racemate is placed in contact with a thin membrane barrier. Theconcentration or pressure differential causes preferential transportacross the membrane barrier. Separation occurs as a result of thenon-racemic chiral nature of the membrane which allows only oneenantiomer of the racemate to pass through. Chiral chromatography,including simulated moving bed chromatography, is used in oneembodiment. A wide variety of chiral stationary phases are commerciallyavailable.

Since superoxide anion is an available and continuously-formedby-product generated through normal metabolic processes, and since itselimination is mediated either by dismutation by the enzyme SOD or viaits reaction with NO to form the potentially hazardous peroxynitrite,without being limited to any theory, the compounds are believed to becapable of simultaneously and favorably affecting both components; theNO and O₂ ⁻. By virtue of the β-agonist activity, NO donation andsuperoxide scavenging properties being simultaneously delivered by thesame molecule, the compounds of the present invention can increase thelevel of NO and reduce levels of superoxide thereby avoiding high levelsof peroxynitrite and oxidant metabolites thereof and consequentlyincreasing the effectiveness of the respiratory active agent.

In one embodiment, compounds of Formula 1, below, are provided:

wherein R¹ is selected from the group consisting of —OH, —ONO, —ONO₂,—SNO, and —NONOate;

R² is alkyl or substituted alkyl, e.g., an optionally substitutedstraight or branched chain C₁-C₁₅ alkyl or optionally substituted C₅-C₈cyclic alkyl;

or optionally R² is a an antioxidant group, such as an ROS scavenger;

or optionally R² is a group comprising an antioxidant, such as an ROSscavenger;

or optionally R² is a group comprising an NO donor and an antioxidant,such as an ROS scavenger, wherein optionally R² comprises a dithiol;

or optionally R² is a straight chain, branched or cyclic alkyl groupsubstituted with an antioxidant, such as an ROS scavenger, such as anitroxide free radical; or a straight chain, branched or cyclic alkylgroup substituted with an NO donor; or a straight chain, branched orcyclic alkyl group substituted with an NO donor and an antioxidant, suchas an ROS scavenger; or a straight chain, branched or cyclic alkyl groupsubstituted with an NO donor and at least one aryl or heteroaryl group;or a cyclic alkyl group substituted with a straight or branched chainalkyl group and/or an NO donor;

or optionally R² is, or is a group comprising: an alkenyl group;substituted alkenyl group; aryl group; or substituted aryl group, wherethe aryl or alkenyl group is substituted with, for example, —OH, —NH₂,—NHCHO or an NO donor group;

or optionally R² is a group comprising a dithiol, for example, R² is astraight chain, branched or cyclic alkyl group substituted with adithiol;

R³ and R⁴ are independently selected from the group consisting of —OH,—CH₂OH, —NH₂, and —NHCHO; or R³ and R⁴ together form an antioxidant,such as an ROS scavenger, such as a substituted N-oxide free radical,such as a substituted pyrrolidine N-oxide free radical, substitutedpiperidine N-oxide free radical, substituted oxazolidine N-oxide freeradical, substituted oxazine N-oxide free radical, substituted thiazoleN-oxide free radical, or substituted thiazine N-oxide free radical; orform amino or hydroxy protecting groups (e.g. N-formyl, acetal orketal);

R⁵ is selected from the group consisting of —H, —OH, —CH₂OH, —NH₂,—NHCHO, straight or branched chain C₁-C₁₅ alkyl, or straight or branchedchain C₁-C₁₅ alkoxy; and

wherein, in a preferred embodiment, the compound includes at least oneROS scavenger and at least one NO-donor (for example independentlypresent on any one or more of R¹⁻⁴).

In another embodiment of Formula 1:

R¹ is —ONO₂, or —SNO;

R² is optionally substituted alkyl, e.g., optionally substituted linearor branched chain C₁-C₁₅ alkyl, or optionally substituted C₅-C₈ cyclicalkyl; or optionally substituted aryl, optionally substitutedheteroaryl, a group comprising a substituted pyrrolidine N-oxide freeradical or substituted piperidine N-oxide free radical, or a group thatis, or may be converted in vivo to, a sulfhydryl in its oxidized orreduced form,

or optionally R² is, or is a group comprising: an alkenyl group;substituted alkenyl group; aryl group; or substituted aryl group, wherethe aryl or alkenyl group is substituted with, for example, —OH, —NH₂,—NHCHO or an NO donor group;

wherein R² is optionally substituted with one or more groups such asC₁-C₃ alkyl groups, C₁-C₃ alkoxy groups, —ONO, —ONO₂, —SNO, aryl, andantioxidant groups; and

R³ and R⁴ are independently —OH, —CH₂OH, —NH₂, or —NHCHO; or togetherform an optionally substituted nitroxide radical, such as substitutedpyrrolidine N-oxide free radical, substituted oxazolidine N-oxide freeradical, or substituted piperidine N-oxide free radical or amino orhydroxy protecting groups; and

R⁵ is selected from the group consisting of —H, —OH, —CH₂OH, —NH₂,—NHCHO, straight or branched chain C₁-C₁₅ alkyl, or straight or branchedchain C₁-C₁₅ alkoxy; and

wherein, in a preferred embodiment, the compound includes at least oneROS scavenger and at least one NO donor.

Exemplary ROS scavenger and antioxidant groups include those asdescribed herein such as groups which incorporate the lipoic acidmoiety, or N-oxide free radical moiety, such as for example, substitutedpyrrolidine, piperidine and oxazolidine N-oxide free radicals, andoptionally substituted aryl groups as well as groups which are, or canbe converted in vivo to, sulfhydryl.

Exemplary substituted pyrrolidine N-oxide, substituted oxazine N-oxideor piperidine N-oxide free radicals which can be formed from R³ and R⁴are shown in Formulas 1a-1e below, where R¹, R² and R⁵ are for exampleas described herein: Thiazole, thiazine and oxazolidine N-oxide freeradical analogues of Formulae 1a-1e are also contemplated.

In some embodiments, compounds of Formula 2 are provided, as shownbelow:

wherein R¹ is selected from the group consisting of —OH, —ONO, —ONO₂,—SNO, and —NONOate;

R² is alkyl or substituted alkyl, e.g., an optionally substitutedstraight or branched chain C₁-C₁₅ alkyl or optionally substituted C₅-C₈cyclic alkyl;

or optionally R² is an antioxidant group, such as an ROS scavenger;

or optionally R² is a group comprising an antioxidant, such as an ROSscavenger; or a group comprising an NO donor and an antioxidant, such asan ROS scavenger, wherein optionally R² comprises a dithiol;

or optionally R² is a straight chain, branched or cyclic alkyl groupsubstituted with an antioxidant, such as an ROS scavenger, such as anitroxide free radical; or a straight chain, branched or cyclic alkylgroup substituted with an NO donor; or a straight chain, branched orcyclic alkyl group substituted with an NO donor and an antioxidant, suchas an ROS scavenger; or a cyclic alkyl group substituted with a straightor branched chain alkyl group and/or an NO donor; or a straight chain,branched or cyclic alkyl group substituted with an NO donor and at leastone aryl or heteroaryl group; or a cyclic alkyl group substituted with astraight or branched chain alkyl group and/or an NO donor;

or optionally R² is, or is a group comprising: an alkenyl group;substituted alkenyl group; aryl group; or substituted aryl group, wherethe aryl or alkenyl group is substituted with, for example, —OH, —NH₂,—NHCHO or an NO donor group;

or optionally R² is a group comprising a dithiol, such as a straightchain, branched or cyclic alkyl group substituted with a dithiol;

R³ and R⁴ are independently selected from the group consisting of —OH,—CH₂OH, —NH₂, and —NHCHO; or R³ and R⁴ together form an antioxidant,such as an ROS scavenger, such as a substituted N-oxide free radical,such as a substituted pyrrolidine N-oxide free radical, substitutedpiperidine N-oxide free radical, substituted oxazolidine N-oxide freeradical, substituted oxazine N-oxide free radical, substituted thiazoleN-oxide free radical, or substituted thiazine N-oxide free radical; orform amino or hydroxy protecting groups (e.g. N-formyl, acetal orketal);

wherein the N-oxide free radical may be substituted with one or moregroups independently selected C₁-C₃ alkyl groups and C₁-C₃ alkoxygroups;

wherein, in a preferred embodiment, the compound includes at least oneROS scavenger and at least one NO donor.

In certain embodiments of Formula 2,

R¹ is selected from the group consisting of —OH, —ONO, —ONO₂, —SNO, and—NONOate;

R² is an optionally substituted alkyl such as an optionally substitutedstraight or branched chain C₁-C₁₅ alkyl group or optionally substitutedC₅-C₈ cyclic alkyl group, a group comprising an ROS scavenger, or agroup comprising an ROS scavenger and/or an NO-donor;

wherein R² may be optionally substituted with, e.g., alkyl such as C₁-C₃alkyl groups; or alkoxy, such as C₁-C₃ alkoxy groups; aryl; NO donorgroups; or a lipoic acid moiety;

R³ and R⁴ are independently selected from the group consisting of —OH,—CH₂OH, —NH₂, and —NHCHO; or R³ and R⁴ together form a substitutedN-oxide free radical, such as a substituted pyrrolidine N-oxide freeradical, substituted oxazolidine N-oxide free radical, or a substitutedpiperidine N-oxide free radical;

wherein the N-oxide free radical may be substituted with one or moregroups independently selected C₁-C₃ alkyl groups and C₁-C₃ alkoxygroups;

wherein, in a preferred embodiment, the compound includes at least oneROS scavenger and at least one NO donor.

In certain embodiments of Formula 1 or Formula 2 at least one of R¹, R²,R³ or R⁴ comprises at least one ROS scavenger; and

at least one of R¹, R², R³ and R⁴ comprises at least one NO donor.

In another embodiment of Formula 1 or 2:

R¹ is —ONO₂, or —SNO;

R² is optionally substituted alkyl, optionally substituted aryl,optionally substituted heteroaryl, substituted pyrrolidine N-oxide freeradical, substituted oxazolidine free radical or substituted piperidineN-oxide free radical, or a group that is, or may be converted in vivoto, a sulfhydryl in its oxidized or reduced form, for example a dithiol;

-   -   wherein R² is optionally substituted with one or more groups        such as C₁-C₃ alkyl groups, C₁-C₃ alkoxy groups, —ONO, —ONO₂,        —SNO, aryl, and antioxidant groups; and

R³ and R⁴ are independently OH, CH₂OH, NH₂, or —NHCHO; or together forma group comprising a substituted nitroxide radical or amino or hydroxyprotecting groups;

-   -   wherein the nitroxide may be substituted with one or more        independently selected C₁-C₃ alkyl groups or C₁-C₃ alkoxy        groups;

wherein, in a preferred embodiment, the compound includes at least oneROS scavenger and at least one NO donor.

In certain embodiments of Formula 1 and Formula 2, R¹, R², R³, R⁴ andR⁵, are one of the embodiments as defined herein, and the compoundincludes at least one ROS scavenger and at least one NO donor.

In certain embodiments, R² comprises one or more substitutedpyrrolidine, oxazolidine, oxazine, thiazole, thiazine or piperidineN-oxide free radicals, aryl moieties, or heteroaryl moieties, whereinthe N-oxide, aryl and heteroaryl moieties may be independentlysubstituted by one or more of the following groups, including one ormore C₁-C₁₅ alkyl groups, C₅-C₈ cyclic alkyl groups, C₁-C₁₅ alkoxygroups, hydroxy groups, amino groups (NH₂), —NHCHO groups, —CH₂OHgroups, or NO donor groups, such as —ONO, —ONO₂ or —SNO.

In another embodiment of Formula 1 or Formula 2, R² may be or comprisean antioxidant such as an ROS scavenger which optionally furtherincludes an NO donor. The antioxidant group is e.g., an alkenyl group;aryl group; substituted aryl group, where the aryl group is substitutedwith, for example, —OH, —NH₂, —NHCHO or a NO donor group. In anotherembodiment, the antioxidant group is, e.g., heteroaryl, dithiol,substituted pyrrolidine, substituted oxazolidine, substituted oxazine,substituted thiazole, substituted thiazine or substituted piperidineN-oxide free radical moiety, or a lipoic acid moiety. In someembodiments, the lipoic acid moiety is substituted with one or more —OH,—ONO, —ONO₂, or —SNO groups. In particular embodiments, R² is analiphatic (CH₂)_(p) linker further attached to a reactive oxygen speciesscavenger group, wherein p is an integer between 2 and 12, e.g. 2-6,6-8, 8-12, and wherein the linker is optionally substituted by one ormore of the following groups such as alkyl including C₁-C₁₅ alkyl,C₁-C₁₅ alkoxy, hydroxy, amino, —NHCHO, —CH₂OH, aryl or an NO donor groupsuch as —ONO, —ONO₂, and —SNO. R² also may be an ROS scavenger and NOdonor group connected by an aliphatic linker (CH₁)_(p).

In one embodiment of Formula 1 and Formula 2:

at least one of R¹, R², R³ or R⁴ comprises at least one ROS scavengergroup;at least one of R¹, R², R³ or R⁴ comprises at least one NO donor.

In another embodiment, where there is more than one NO donor group, theNO donor groups are the same.

In one embodiment of Formula 1,

R¹ is selected from the group consisting of —OH, —ONO₂, and —SNO;

R² is optionally substituted alkyl, such as linear, or branched chainC₁-C₁₅ alkyl or C₅-C₈ cyclic alkyl; including, an alkyl groupsubstituted with a substituted pyrrolidine, substituted oxazolidine orsubstituted piperidine N-oxide free radical; an alkyl group substitutedwith one or more aryl or heteroaryl groups; or an alkyl groupsubstituted with a group that is, or may be converted in vivo to, asulfhydryl in its oxidized or reduced form, such as, for example, adithiol or a lipoic acid moiety;

wherein R² may be additionally optionally substituted with one or moreindependently selected groups including C₁-C₃ alkyl groups, C₁-C₃ alkoxygroups, ONO₂, or —SNO;

R³ and R⁴ are independently —OH; CH₂OH; —NH₂; —NHCHO; or R³ and R⁴together form a substituted pyrrolidine, substituted oxazolidine,substituted oxazine, substituted thiazole, substituted thiazine orsubstituted piperidine N-oxide free radical, or together form an aminoor hydroxy protecting group (e.g. N-formyl, acetal or ketal); whereinthe N-oxide free radical is optionally substituted with one or moregroups independently selected from C₁-C₃ alkyl groups and C₁-C₃ alkoxygroups; and

R⁵ is selected from the group consisting of —H, —OH, —CH₂OH, —NH₂,—NHCHO, and straight or branched chain C₁-C₃ alkyl, straight or branchedchain C₁-C₃ alkoxy.

In another embodiment of Formula 1,

R¹ is —ONO₂ or —SNO;

R² is optionally substituted alkyl, such as linear or branched chainC₁-C₁₅ alkyl, or optionally substituted C₅-C₈ cyclic alkyl; asubstituted pyrrolidine, substituted oxazolidine or substitutedpiperidine N-oxide free radical; an alkyl group substituted with an arylor heteroaryl group; an alkyl group substituted with an N-oxide freeradical, such as substituted pyrrolidine, substituted oxazolidine orsubstituted piperidine N-oxide free radical; or a group that is, or maybe converted in vivo to, a sulfhydryl in its oxidized or reduced form,such as a dithiol or lipoic acid moiety;

wherein R² may be optionally substituted with —ONO₂, or —SNO;

R³ and R⁴ together form a substituted pyrrolidine, or substitutedpiperidine nitroxide free radical, or form amino or hydroxy protectinggroups (e.g. N-formyl, acetal or ketal); and

R⁵ is selected from the group consisting of H, —OH, —CH₂OH, —NH₂,—NHCHO, and straight or branched chain C₁-C₃ alkyl, straight or branchedchain C₁-C₃ alkoxy.

In another embodiment of Formula 1,

R¹ is —OH, —ONO₂, or —SNO;

-   -   R² is or comprises an optionally substituted linear or branched        chain C₁-C₁₅ alkyl group or an optionally substituted C₅-C₈        cyclic alkyl group; a substituted pyrrolidine or substituted        piperidine N-oxide free radical; an alkyl group substituted with        one or more aryl or heteroaryl group; or an alkyl group        substituted with a group that is or may be converted in vivo to        a sulfhydryl in its oxidized or reduced form;    -   wherein R² may be optionally substituted with one or more groups    -   such as C₁-C₃ alkyl groups, C₁-C₃ alkoxy groups, —ONO, —ONO₂, or        —SNO;    -   R³ and R⁴ are —OH, —CH₂OH, —NCHOH, or together form a        substituted pyrrolidine or substituted piperidine N-oxide free        radical; and    -   R⁵ is selected from the group consisting of —H, —OH, —CH₂OH,        —NH₂, —NHCHO, straight or branched chain C₁-C₃ alkyl, and        straight or branched chain C₁-C₃ alkoxy; and wherein in one        preferred embodiment R⁵ is —H.

In one embodiment, R² is:

wherein m is 1-6 and R⁸ and R⁹ are independently C₁-C₃ alkyl or —H.

In another embodiment of Formula 1,

-   -   R¹ is —ONO₂ or —SNO;    -   R² is optionally substituted alkyl such as linear or branched        C₁-C₁₅ alkyl or optionally substituted C₅-C₈ cyclic alkyl; a        group comprising substituted pyrrolidine or substituted        piperidine N-oxide free radical;    -   or an alkyl group substituted with one or more aryl, heteroaryl,        or dithiol group;    -   R³ and R⁴ together form a substituted pyrrolidine N-oxide free        radical or a piperidine N-oxide free radical; and    -   R⁵ is —H.

In another embodiment of Formula 1,

-   -   R¹ is —ONO₂;    -   R² is optionally substituted alkyl such as linear or branched        chain C₁-C₁₅ alkyl; an alkyl group substituted with a        substituted pyrrolidine or substituted piperidine N-oxide free        radical; or an alkyl group substituted with one or more aryl,        heteroaryl or dithiol groups;    -   R³ and R⁴ together form a substituted pyrrolidine or piperidine        N-oxide free radical; and    -   R⁵ is —H.

In some embodiments of Formula 1 or 2, R² is alkyl such as t-butyl or ischosen from the following:

Examples of multifunctional β-agonist compounds may be found in FIGS. 1and 2. Examples of syntheses of such compounds may be found in Examples1-10 and in the description herein.

In compounds of Formula 1 and 2, R¹ is selected from the groupconsisting of —OH, —ONO, —ONO₂, —SNO, and —NONOate. In a preferredembodiment, R¹ is selected from the group consisting of —OH, —ONO₂, and—SNO. In another preferred embodiment, R¹ is —ONO₂, or —SNO. In anotherpreferred embodiment, R¹ is —ONO₂.

In compounds of Formula 1 and 2, in a preferred embodiment, R² is alkylor substituted alkyl, e.g., an optionally substituted straight orbranched chain C₁-C₁₅ alkyl; a group comprising an antioxidant, such asan ROS scavenger; or a group comprising an NO donor and an antioxidant,such as an ROS scavenger, wherein optionally R² comprises a dithiol.

In another preferred embodiment, R² is optionally substituted alkyl,e.g., optionally substituted linear or branched chain C₁-C₁₅ alkyl,optionally substituted C₅-C₈ cyclic alkyl, optionally substituted aryl,optionally substituted heteroaryl, a group comprising a substitutedpyrrolidine N-oxide free radical or substituted piperidine N-oxide freeradical, or a group that is, or may be converted in vivo to, asulfhydryl in its oxidized or reduced form, wherein R² is optionallysubstituted with one or more groups such as C₁-C₃ alkyl groups, C₁-C₃alkoxy groups, —ONO, —ONO₂, —SNO, and an antioxidant group.

In another preferred embodiment, R² is an optionally substituted alkyl,such as an optionally substituted straight or branched chain C₁-C₁₅alkyl group, or optionally substituted C₅-C₈ cyclic alkyl group; a groupcomprising an ROS scavenger; or a group comprising an ROS scavengerand/or an NO-donor; wherein R² may be optionally substituted with, e.g.,alkyl, such as C₁-C₃ alkyl groups or alkoxy, such as C₁-C₃ alkoxygroups.

In another preferred embodiment, R² is optionally substituted alkyl,optionally substituted aryl, optionally substituted heteroaryl, asubstituted pyrrolidine N-oxide free radical or substituted piperidineN-oxide free radical, or a group that is, or may be converted in vivoto, a sulfhydryl in its oxidized or reduced form; wherein R² may beoptionally substituted with one or more groups independently selectedfrom C₁-C₃ alkyl groups, C₁-C₃ alkoxy groups, —ONO, —ONO₂, or —SNO.

In another preferred embodiment, R² comprises one or more substitutedpyrrolidine, oxazolidine, oxazine, thiazole, thiazine or piperidineN-oxide free radicals, aryl moieties, or heteroaryl moieties, whereinthe N-oxide, aryl and heteroaryl moieties may be independentlysubstituted by one or more of the following groups, including one ormore C₁-C₁₅ alkyl groups, C₅-C₈ cyclic alkyl groups, C₁-C₁₅ alkoxygroups, hydroxy groups, amino groups (NH₂), —NHCHO groups, —CH₂OHgroups, or NO donor groups, such as —ONO, —ONO₂ or —SNO.

In another preferred embodiment, R² comprises an antioxidant such as anROS scavenger and optionally further comprises an NO donor. Theantioxidant group is e.g., aryl, heteroaryl, dithiol, substitutedpyrrolidine oxazolidine, oxazine, thiazole, thiazine or piperidineN-oxide free radical moiety, or a lipoic acid moiety. In someembodiments, the lipoic acid moiety is substituted with one or more —OH,—ONO, —ONO₂, or —SNO groups. In particular embodiments, R² is analiphatic (CH₂)_(p) linker further comprising a reactive oxygen speciesscavenger group, wherein p is an integer between 2 and 12, e.g. 2-6,6-8, 8-12, and wherein the linker is optionally substituted by one ormore of the following groups such as alkyl including C₁-C₈ alkyl, C₁-C₉alkoxy, hydroxy, amino, —NHCHO, —CH₂OH, or an NO donor group such as—ONO, —ONO₂, and —SNO. R² also may comprise an ROS scavenger and NOdonor group connected by an aliphatic linker (CH₂)_(p).

In another preferred embodiment, R² is optionally substituted alkyl,such as linear, or branched chain C₁-C₁₅ alkyl or C₅-C₈ cyclic alkyl; agroup comprising a substituted pyrrolidine or substituted piperidineN-oxide free radical; a group comprising an aryl, heteroaryl group; orcomprises a group that is, or may be converted in vivo to, a sulfhydrylin its oxidized or reduced form; wherein R² is optionally substitutedwith one or more independently selected groups including C₁-C₃ alkylgroups, C₁-C₃ alkoxy groups, —ONO₂, or —SNO.

In another preferred embodiment, R² is optionally substituted alkyl,such as linear or branched chain C₁-C₁₅ alkyl, or a group comprising anoptionally substituted C₅-C₈ cyclic alkyl; a substituted pyrrolidine orsubstituted piperidine N-oxide free radical; a groups comprising an arylor heteroaryl group; or a group that is, or may be converted in vivo to,a sulfhydryl in its oxidized or reduced form; wherein R² may beoptionally substituted with —ONO₂, or —SNO.

In another preferred embodiment, R² comprises an optionally substitutedlinear or branched chain C₁-C₁₅ alkyl group or an optionally substitutedC₅-C₈ cyclic alkyl groups; a substituted pyrrolidine or piperidineN-oxide free radical; a group comprising an aryl or heteroaryl group; ora group that is or may be converted in vivo to a sulfhydryl in itsoxidized or reduced form; wherein R² may be optionally substituted withone or more groups such as C₁-C₃ alkyl groups, C₁-C₃ alkoxy groups,—ONO, —ONO₂, or —SNO.

In another preferred embodiment, R² is:

wherein m is 1-6 and R⁸ and R⁹ are independently C₁-C₃ alkyl or H.

In another preferred embodiment, R² is optionally substituted alkyl suchas linear or branched C₁-C₁₅ alkyl or optionally substituted C₅-C₈cyclic alkyl; a group comprising substituted pyrrolidine or substitutedpiperidine N-oxide free radical; or a group comprising an aryl,heteroaryl, or dithiol.

In another preferred embodiment, R² is optionally substituted alkyl suchas linear or branched chain C₁-C₁₅ alkyl; a group comprising asubstituted pyrrolidine or substituted piperidine N-oxide free radical;or a group comprising an aryl, heteroaryl or dithiol.

In compounds of Formula 1 and 2, R³ and R⁴ may be independently selectedfrom the group consisting of —OH, —CH₂OH, NH₂, and —NHCHO; or R³ and R⁴together form an antioxidant, such as an ROS scavenger, such as asubstituted N-oxide free radical, such as a substituted pyrrolidineN-oxide free radical, substituted piperidine N-oxide free radical,substituted oxazolidine N-oxide free radical, substituted oxazineN-oxide free radical, substituted thiazole N-oxide free radical, orsubstituted thiazine N-oxide free radical.

In a preferred embodiment, R³ and R⁴ are independently —OH, —CH₂OH,—NH₂, or —NHCHO; or together form an optionally substituted nitroxideradical, such as substituted pyrrolidine N-oxide free radical orsubstituted piperidine N-oxide free radical or amino or hydroxyprotecting groups.

In another preferred embodiment, R³ and R⁴ are independently selectedfrom the group consisting of —OH, —CH₂OH, —NH₂, and —NHCHO; or R³ and R⁴together form a substituted N-oxide free radical, such as a substitutedpyrrolidine N-oxide free radical, or a substituted piperidine N-oxidefree radical; wherein the N-oxide free radical may be substituted withone or more groups independently selected C₁-C₃ alkyl groups and C₁-C₃alkoxy groups.

In another preferred embodiment, R³ and R⁴ are independently —OH,—CH₂OH, —NH₂, or —NHCHO; or together form a group comprising asubstituted nitroxide radical or amino or hydroxy protecting groups;wherein the nitroxide may be substituted with one or more independentlyselected C₁-C₃ alkyl groups or C₁-C₃ alkoxy groups.

In another preferred embodiment, R³ and R⁴ are independently —OH;—CH₂OH; —NH₂; —NHCHO; or R³ and R⁴ together form a substitutedpyrrolidine or substituted pyrrolidine substituted oxazolidine,substituted oxazine, substituted thiazole, substituted thiazine orsubstituted piperidine N-oxide free radical, or together form an aminoor hydroxy protecting group (e.g. N-formyl, acetal or ketal); whereinthe N-oxide free radical is optionally substituted with one or moregroups independently selected from C₁-C₃ alkyl groups and C₁-C₃ alkoxygroups.

In another preferred embodiment, R³ and R⁴ together form a substitutedpyrrolidine or piperidine nitroxide radical, or form amino or hydroxyprotecting groups (e.g. N-formyl, acetal or ketal).

In another preferred embodiment, R³ and R⁴ are OH, CH₂OH, —NCHOH, ortogether form a substituted pyrrolidine or substituted piperidineN-oxide free radical.

In another preferred embodiment, R³ and R⁴ together form a substitutedpyrrolidine N-oxide free radical or a substituted piperidine N-oxidefree radical.

In compounds of Formula 1, R⁵ is selected from the group consisting of—H, —OH, —CH₂OH, —NH₂, —NHCHO, straight or branched chain C₁-C₁₅ alkyl,or straight or branched chain C₁-C₁₅ alkoxy.

In another preferred embodiment, R⁵ is selected from the groupconsisting of —H, —OH, —CH₂OH, —NH₂, —NHCHO, and straight or branchedchain C₁-C₃ alkyl, straight or branched chain C₁-C₃ alkoxy.

In another preferred embodiment, R⁵ is —H.

In compounds of Formula 1 and 2, in a preferred embodiment, at least oneof R¹, R², R³ or R⁴ comprises at least one ROS scavenger.

In another preferred embodiment, at least one of R¹, R², R³ or R⁴comprises at least one NO donor.

In another preferred embodiment, the compound includes at least one ROSscavenger and at least one NO donor.

In another preferred embodiment, each of R¹, R², R³, R⁴ and R⁵ areselected the embodiments as defined herein, and the compound includes atleast one ROS scavenger and at least one NO donor.

In another preferred embodiment, each of R¹, R², R³, R⁴ and R⁵ areselected from the embodiments as defined herein.

In another preferred embodiment, where there is more than one NO donor,the NO donor groups are the same.

Synthesis of Multifunctional β-Agonist Compounds

Multifunctional β-agonist compounds may be synthesized as describedherein using methods available in the art and standard techniques inorganic chemistry, as described, for example, in March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition(2000) M. B. Smith & J. March, John Wiley & Sons, New York, N.Y.;Organic Chemistry 6^(th) Ed. (1992) R. Morrison & R. Boyd, BenjaminCummings, San Francisco. Synthetic methods such as these are known inthe art and are further described in Hett et al. (1994) TetrahedronLetters 35(50):9375-9378 and Hett et al. (1997) Tetrahedron Letters38(7):1125-1128.

Examples of multifunctional β-agonist compounds are compounds 1-10 shownin FIGS. 1 and 2, while the details of their synthesis are described inExamples 1-10.

The general approach for synthesis of compounds of the general formula Iis outlined in Scheme 1 below:

The starting materials, H₂NR², are chiral or non-chiral amines, usuallyin the N-benzylated form. Synthetic methods such as these are known inthe art and are further described in Hett et al. (1994) TetrahedronLetters 35(50):9375-9378 and Hett et al. (1997) Tetrahedron Letters38(7):1125-1128.

A more detailed reaction scheme for the synthesis of the non-chiral andchiral epoxides is shown below in Scheme 2.

In cases where the aryl ring of the β-agonist constitutes itself a ROSscavenger group (e.g., 1,1,3,3-tetramethylisoindolin-2-yloxyl freeradical), the chiral/non-chiral epoxides are synthesized as shown indetail in Scheme 3 below,

Scheme 3, above, depicts the synthesis of a non-chiral epoxideincorporating a stable nitroxide radical containing a 5-membered ring(PROXYL moiety). Methods of bromination of acetophenone derivatives areavailable in the art, such as bromination with Br₂ and bromination withpyrrolidinone hydrotribromide.

Respiratory Disorders

The present invention provides the compounds of the present inventionfor use in therapy.

The present invention further provides use of the compounds of thepresent invention in the manufacture of a medicament for the treatmentof respiratory disorders.

The multifunctional β-agonist compounds are useful in treating a varietyof respiratory disorders. Respiratory disorders include asthma, chronicbronchitis, bronchiectasis and emphysema, Chronic Obstructive PulmonaryDiseases (COPDs) or Chronic Obstructive Airway Disease (COADs).

COPDs are often characterized as being accompanied by chronic orrecurrent obstruction to air flow within the lung. Increased resistanceto air flow may result from narrowing or restriction of an airway at anylevel, including partial or complete obstruction from the trachea andlarger bronchi to the terminal and respiratory bronchioles.

Another major class of pulmonary or respiratory diseases are oftenreferred to as restrictive diseases, which maybe characterized byreduced expansion of lung parenchyma, with a reduced total lungcapacity. Many pathologic conditions associated with respiratorydisorders have both obstructive and restrictive components (Cotran etal., “Robbins Pathologic Basis of Disease” 4th Ed. 1989, W.B. SaundersCo., Philadelphia, Pa., pp 755-797).

Asthma may be characterized as an obstructive respiratory disordercharacterized by increased responsiveness of the airway to variousstimuli, which may potentiate spasmic constriction of the bronchialairways. Asthma can occur secondarily to a variety of stimuli (Cotran etal., “Robbins Pathologic Basis of Disease” 4th Ed. 1989, W.B. SaundersCo., Philadelphia, Pa., pp 755-797). Chronic asthma can also beconsidered to be predominantly an inflammatory disease with associatedbronchospasm. The degree of reactivity and narrowing of the bronchi inresponse to stimuli is greater in asthmatics than in normal individuals.Persistent inflammation may be responsible for the bronchialhyperreactivity or airway hyperresponsiveness (AHR). Mucosal edema,mucus plugging and hypersecretion XX can be also present; and pulmonaryparenchyma can be common. Asthmatics manifesting such imbalance usuallyhave hyperactive bronchi and, even without symptoms, bronchoconstrictionmay be present. Overt asthma attacks may occur when such individuals aresubjected to various stresses, such as viral respiratory infection,exercise, emotional upset, nonspecific factors (e.g., changes inbarometric pressure or temperature), inhalation of cold air or irritants(e.g., gasoline fumes, fresh paint and noxious odors, or cigarettesmoke), exposure to specific allergens, and ingestion of aspirin orsulfites in sensitive individuals. Asthma is often characterized as“extrinsic” or “allergic”, where the asthmatic episode is precipitatedby allergens (e.g., most commonly airborne pollens and molds, housedust, animal danders) or “nonallergic” or “intrinsic”, where symptomaticepisodes seem to be triggered by non-allergenic factors (e.g. infection,irritants, emotional factors). In some individuals both allergenic andnon-allergenic factors may be significant.

The compounds described herein can be used in the treatment of intrinsicand extrinsic asthma. They are especially applicable to the treatment ofallergic or atopic (e.g. IgE-mediated) asthma or non-atopic asthma, aswell as exercise induced asthma, occupational asthma, asthma inducedfollowing bacterial infection or drug, e.g. aspirin, ingestion and othernon-allergic asthmas. The multifunctional β-agonist compounds may alsobe used in the treatment and/or prophylaxis of respiratory conditionssuch as chronic obstructive pulmonary or airways disease (COPD or COAD),chronic bronchitis, emphysema, respiratory distress syndrome (in childor adult), pneumonia, bronchial hyperreactivity, bronchiectasis, andairway hyperresponsiveness (AHR).

Asthma is often categorized as atopic (allergic), nonreaginic (whereprecipitating factor is a respiratory infection), pharmacologic (e.g.aspirin-sensitive or other drug sensitivity), occupational (e.g.chemical challenge from environmental stimuli), allergicbronchopolumonary aspergillosis (antigen challenge (e.g. spores))(Cotran et al., “Robbins Pathologic Basis of Disease” 4th Ed. 1989, W.B.Saunders Co., Philadelphia, Pa., pp 755-797). The multifunctionalβ-agonist compounds discussed herein may be used in the treatment ofeach of these conditions or where combinations of factors areresponsible for the clinical manifestation of the disorder.

Chronic bronchitis is a condition often associated with prolongedexposure to bronchial irritants and accompanied by mucus hypersecretionand certain structural changes in the bronchi. Usually associated withcigarette smoking, it is characterized clinically by chronic productivecough. Chronic obstructive bronchitis is often characterized as chronicbronchitis associated with extensive alterations of the small airwaysleading to clinically significant airways obstruction (Cotran et al.,“Robbins Pathologic Basis of Disease” 4th Ed. 1989, W.B. Saunders Co.,Philadelphia, Pa., pp 755-797).

The present invention is especially applicable in the treatment ofrespiratory conditions including, but not limited to, the respiratorydisorders disclosed herein. As used herein, and as well-understood inthe art, “treatment” is an approach for obtaining beneficial or desiredresults, including clinical results. For purposes of this invention,beneficial or desired clinical results can include one or more, but arenot limited to, alleviation or amelioration of one or more symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, preventing spread of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable.

The present invention is applicable in the treatment of severe acuterespiratory syndrome (SARS). The current treatment protocol for this,most probably viral, disorder, is mainly supportive, and entailsadministration of oxygen and β-agonists.

Preferred are compounds that are potent and can be administered locallyat very low doses, thus avoiding systemic adverse effects. Compoundsalso are preferred that possess both cAMP and cGMP stimulating activityvia simultaneous activation of adenylyl cyclase and guanylyl cyclase,respectively. Also preferred are compounds with potent antioxidantcharacteristics and concurrent potent mucolytic properties. Themultifunctional β-agonist compounds with anti-ROS activity and NO donorproperties can exert a significant impact on the severity, control, andthe natural course of respiratory diseases involving oxidative stressand free radical injury. Because of their multi-mechanistic actions,tolerance to their broncho-protective effect can preferably be avoided.The absence of tolerance can avoid the necessity of medium- to high-dosesteroid therapy. The development of tolerance is disadvantageous sincewhen administering a composition or drug in repeated dosage or over aperiod of time, the amount of the composition or the frequency ofadministration of the drug or composition given to the patient must beincreased in order to achieve the same effect as the lower dosage givenat an earlier time point in the course of treatment.

Formulations and Dosage

The compounds can be provided in a variety of formulations and dosages.The compounds may be provided in a pharmaceutically acceptable formand/or in a salt form.

In one embodiment, the compounds are provided as non-toxicpharmaceutically acceptable salts. Suitable pharmaceutically acceptablesalts of the compounds of this invention include acid addition saltssuch as those formed with hydrochloric acid, fumaric acid,p-toluenesulphonic acid, maleic acid, succinic acid, acetic acid, citricacid, tartaric acid, carbonic acid or phosphoric acid. Salts of aminegroups may also comprise quaternary ammonium salts in which the aminonitrogen atom carries a suitable organic group such as an alkyl,alkenyl, alkynyl or aralkyl moiety. Furthermore, where the compounds ofthe invention carry an acidic moiety, suitable pharmaceuticallyacceptable salts thereof may include metal salts such as alkali metalsalts, e.g. sodium or potassium salts; and alkaline earth metal salts,e.g. calcium or magnesium salts.

The pharmaceutically acceptable salts of the present invention may beformed by conventional means, such as by reacting the free base form ofthe product with one or more equivalents of the appropriate acid in asolvent or medium in which the salt is insoluble, or in a solvent suchas water which is removed in vacuo or by freeze drying or by exchangingthe anions of an existing salt for another anion on a suitable ionexchange resin.

The present invention includes within its scope solvates of themultifunctional β-agonist compounds and salts thereof, for example,hydrates.

The multifunctional β-agonist compounds may have one or more asymmetriccentres, and may accordingly exist both as enantiomers and asdiastereoisomers. It is to be understood that all such isomers andmixtures thereof are encompassed within the scope of the presentinvention.

The multifunctional β-agonist compounds may be administered by oral,parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV,intracisternal injection or infusion, subcutaneous injection, orimplant), buccal, by inhalation spray, nasal, vaginal, rectal,sublingual, or topical routes of administration and may be formulated,alone or together, in suitable dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvants,excipients and vehicles appropriate for each route of administration. Inaddition to the treatment of warm-blooded animals such as mice, rats,horses, cattle, sheep, dogs, cats, monkeys, etc., the compounds of theinvention may be effective in humans.

The pharmaceutical compositions for the administration of themultifunctional β-agonist compounds may conveniently be presented indosage unit form and may be prepared by any of the methods well known inthe art of pharmacy. The pharmaceutical compositions can be, forexample, prepared by uniformly and intimately bringing the activeingredient into association with a liquid carrier or a finely dividedsolid carrier or both, and then, if necessary, shaping the product intothe desired formulation. In the pharmaceutical composition the activeobject compound is included in an amount sufficient to produce thedesired therapeutic effect.

The pharmaceutical compositions containing the multifunctional β-agonistcompound as active ingredient may be in a form suitable for oral use,for example, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsions, hard or soft capsules, orsyrups or elixirs. Compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. Tabletscontain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated by the techniques described in the U.S. Pat. Nos. 4,256,108;4,166,452; and 4,265,874 to form osmotic therapeutic tablets for controlrelease. The pharmaceutical compositions of the invention may also be inthe form of oil-in-water emulsions.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Themultifunctional β-agonist compounds may also be administered in the formof suppositories for rectal administration of the drug.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the multifunctional β-agonist compounds may beemployed.

According to the present invention, multifunctional β-agonist compoundscan be delivered by any of a variety of inhalation devices and methodsknown in the art, including, for example: U.S. Pat. No. 6,241,969; U.S.Pat. No. 6,060,069; U.S. Pat. No. 6,238,647; Hughes et al., U.S. Pat.No. 6,335,316; Rubsamen; U.S. Pat. No. 5,364,838; Rubsamen, U.S. Pat.No. 5,672,581; Platz, et al., WO96/32149; Patton, et al., WO95/24183;Johnson, et al., U.S. Pat. No. 5,654,007; Goodman, et al., U.S. Pat. No.5,404,871; Rubsamen, et al., U.S. Pat. No. 5,672,581; Gonda, et al.,U.S. Pat. No. 5,743,250; Rubsamen, U.S. Pat. No. 5,419,315; Rubsamen, etal., U.S. Pat. No. 5,558,085; Gonda, et al., WO98/33480; Rubsamen, U.S.Pat. No. 5,364,833; Laube, et al., U.S. Pat. No. 5,320,094; Eljamal, etal. U.S. Pat. No. 5,780,014; Backstrom, et al., U.S. Pat. No. 5,658,878;Backstrom, et al., 5,518,998; Backstrom, et al., 5,506,203; Meezan, etal., U.S. Pat. No. 5,661,130; Hodson, et al., U.S. Pat. No. 5,655,523;Schultz, et al., U.S. Pat. No. 5,645,051; Eisele, et al., U.S. Pat. No.5,622,166; Mecikalski, et al., U.S. Pat. No. 5,577,497; Mecikalski, etal., U.S. Pat. No. 5,492,112; Williams, et al., U.S. Pat. No. 5,327,883;Williams, U.S. Pat. No. 5,277,195; U.S. Pat. App. No. 20010041190; U.S.Pat. App. No. 20020006901; and U.S. Pat. App. No. 20020034477.

Included among the devices which may be used to administer particularexamples of the multifunctional β-agonist compounds are those well-knownin the art, such as, metered dose inhalers, liquid nebulizers, drypowder inhalers, sprayers, thermal vaporizers, and the like. Othersuitable technology for administration of particular multifunctionalβ-agonist compounds includes electrohydrodynamic aerosolizers.

The abbreviations “MMAD” and “MMEAD” are well-known in the art, andstand for “mass median aerodynamic diameter” and “mass median equivalentaerodynamic diameter” respectively. The terms as used in the art aresubstantially equivalent. The “aerodynamic equivalent” size of aparticle is the diameter of a unit density sphere which exhibits thesame aerodynamic behavior as the particle, regardless of actual densityor shape. MMAD is usually determined using a cascade impactor, whichmeasures-the particle size as a function of the aerodynamic behavior ofthe particle in a high velocity airstream. The median (50%) particlesize is obtained from a linear regression analysis of the cumulativedistribution data. In one embodiment, the inhalation device deliverssmall particles, e.g., less than about 10 μm MMAD.

In addition, the inhalation device is preferably practical, in the senseof being easy to use, small enough to carry conveniently, capable ofproviding multiple doses, and durable. Some specific examples ofcommercially available inhalation devices are Turbobaler (Astra,Wilmington, Del.), Rotahaler (Glaxo, Research Triangle Park, N.C.),Diskus (Glaxo, Research Triangle Park, N.C.), the Ultravent nebulizer(Mallinckrodt), the Acorn II nebulizer (Marquest Medical Products,Totowa, N.J.) the Ventolin metered dose inhaler (Glaxo, ResearchTriangle Park, N.C.), or the like. In some examples, multifunctionalβ-agonist compounds can be delivered by a dry powder inhaler or asprayer.

As those skilled in the art will recognize, the formulation ofmultifunctional β-agonist compounds, the quantity of the formulationdelivered, and the duration of administration of a single dose depend onthe type of inhalation device employed as well as other factors. Forsome aerosol delivery systems, such as nebulizers, the frequency ofadministration and length of time for which the system is activated willdepend mainly on the concentration of multifunctional β-agonistcompounds in the aerosol. For example, shorter periods of administrationcan be used at higher concentrations of multifunctional β-agonistcompounds in the nebulizer solution. Devices such as metered doseinhalers can produce higher aerosol concentrations, and can be operatedfor shorter periods to deliver the desired amount of multifunctionalβ-agonist compounds in some embodiments. Devices such as dry powderinhalers deliver active agent until a given charge of agent is expelledfrom the device. In this type of inhaler, the amount of multifunctionalβ-agonist compounds in a given quantity of the powder determines thedose delivered in a single administration. The formulation ofmultifunctional β-agonist compound is selected to yield the desiredparticle size in the chosen inhalation device.

Dry powder generation typically employs a method such as a scraper bladeor an air blast to generate particles from a solid formulation ofmultifunctional β-agonist compounds. The particles are generallygenerated in a container and then transported into the lung of a patientvia a carrier air stream. Typically, in current dry powder inhalers, theforce for breaking up the solid and air flow is provided solely by thepatient's inhalation. One suitable dry powder inhaler is the Turbohalermanufactured by Astra (Wilmington, Del.).

Formulations of multifunctional β-agonist compounds for administrationfrom a dry powder inhaler may typically include a finely divided drypowder containing multifunctional β-agonist compounds, but the powdercan also include a bulking agent, buffer, carrier, excipient, anotheradditive, or the like. Additives can be included in a dry powderformulation of multifunctional β-agonist compounds, for example, todilute the powder as required for delivery from the particular powderinhaler, to facilitate processing of the formulation, to provideadvantageous powder properties to the formulation, to facilitatedispersion of the powder from the inhalation device, to stabilize to theformulation (e.g., antioxidants or buffers), to provide taste to theformulation, or the like. Typical additives include mono-, di-, andpolysaccharides; sugar alcohols and other polyols, such as, for example,lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose,sucrose, mannitol, starch, or combinations thereof; surfactants, such assorbitols, diphosphatidyl choline, or lecithin; or the like.

In some embodiments, a spray including multifunctional β-agonistcompounds can be produced by forcing a suspension or solution of aparticular multifunctional β-agonist compound through a nozzle underpressure. The nozzle size and configuration, the applied pressure, andthe liquid feed rate can be chosen to achieve the desired output andparticle size. An electrospray can be produced by an electric field inconnection with a capillary or nozzle feed. Formulations ofmultifunctional β-agonist compounds suitable for use with a sprayer caninclude multifunctional β-agonist compounds in an aqueous solution at aconcentration of about 1 mg to about 20 mg of multifunctional β-agonistcompound per mL of solution. The formulation can include agents such asan excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and/or, zinc. Multifunctional β-agonist compounds can beadministered by a nebulizer, such as jet nebulizer or an ultrasonicnebulizer. Typically, in a jet nebulizer, a compressed air source isused to create a high-velocity air jet through an orifice.

Formulations of multifunctional β-agonist compound suitable for use witha nebulizer, either jet or ultrasonic, typically include multifunctionalβ-agonist compound in an aqueous solution. The formulation can includeagents such as an excipient, a buffer, an isotonicity agent, apreservative, a surfactant, and/or zinc. The formulation can alsoinclude an excipient or agent for stabilization of the multifunctionalβ-agonist compound, such as a buffer, a reducing agent, a bulk protein,or a carbohydrate. Bulk proteins, surfactants, carbohydrates and otheradditives are useful in formulating multifunctional β-agonist compoundsand can be used as described above.

In a metered dose inhaler (MDI), a propellant, multifunctional β-agonistcompound, and any excipients or other additives are contained in acanister as a mixture including a liquefied compressed gas.

The present invention also relates to a pharmaceutical compositionincluding multifunctional β-agonist compounds suitable foradministration by inhalation. According to the invention,multifunctional β-agonist compounds can be used for manufacturing acomposition or medicament, including medicaments suitable foradministration by inhalation. The invention also relates to methods formanufacturing compositions including multifunctional β-agonist compoundsin a form that is suitable for administration, including administrationby inhalation. For example, a dry powder formulation can be manufacturedin several ways, using conventional techniques, such as described in anyof the publications mentioned above and incorporated expressly herein byreference, and, for example, Baker, et al., U.S. Pat. No. 5,700,904, theentire disclosure of which is incorporated expressly herein byreference. Particles in the size range appropriate for maximaldeposition in the lower respiratory tract can be made by micronizing,milling, or the like. And a liquid formulation can be manufactured bydissolving the multifunctional β-agonist compounds in a suitablesolvent, such as water, at an appropriate pH, including buffers or otherexcipients.

As known by those of skill in the art, the preferred dosage ofmultifunctional β-agonist compounds will depend on the age, weight,general health and severity of the respiratory disorder of theindividual being treated. Dosage may also need to be tailored to the sexof the individual and/or the lung capacity of the individual. Dosage mayalso be tailored to individuals suffering from more than one respiratorydisorder or those individuals who have additional conditions whichaffect lung capacity and the ability to breathe normally, for example,emphysema, bronchitis, pneumonia, respiratory infections, etc. Dosage,and frequency of administration of the multifunctional β-agonistcompound will also depend on whether the compounds are formulated fortreatment of acute episodes of the respiratory disorder or for theprophylactic treatment of the disorder. A skilled practitioner will beable to determine the optimal dose for a particular individual. Variousformulations of the compounds and compositions described herein may beadministered according to the variables described above. In particular,formulations for prophylactic treatment of respiratory conditions may beadministered, daily, twice daily, thrice daily or four times dailyand/or upon the occurrence of symptoms associated with the underlyingrespiratory condition. Such symptoms include wheezing, coughing,shortness of breath, tightness or pressure in the chest and the like. Itis contemplated that individuals who are using a prophylacticformulation may on occasion need to administer doses in response toacute episodes of symptoms. Administration includes any of the methodsor routes as described herein.

The compounds as described herein may be administered to an individualin need thereof over a period of time consistent with treatment of therespiratory disorder from which the individual suffers. In the case ofpneumonia or other periodic disorders, the treatment may be discontinuedwhen the individual is no longer affected by the disorder or deemed tobe no longer in need of the treatment by a skilled practitioner.Examples of such time periods include days, weeks or months. Where therespiratory condition is a congenital or chronic disorder such asasthma, emphysema, AHR, COPD and others, it is envisioned that thetreatment with the compounds described herein will be administered for aperiod of weeks, months, years or decades. The methods as describedherein also include the administration of combinations of the compoundsas described herein, or combinations of the compounds described hereinand other drugs used in the treatment of the respiratory disordersdescribed herein or symptoms associated with these disorders.

Drug delivery devices, such as metered inhalation devices, may be usedto deliver the compounds of the invention by inhalation as describedabove.

Also provided are kits for administration of the multifunctionalβ-agonist compound or composition comprising at least onemultifunctional β-agonist compound, that may include a dosage amount ofat least one multifunctional β-agonist compound or a compositioncomprising at least one multifunctional β-agonist compound as disclosedherein. Kits may further comprise suitable packaging and/or instructionsfor use of the compound. Kits may also comprise a means for the deliveryof the at least one multifunctional β-agonist compound or compositionscomprising at least one multifunctional β-agonist compound, such as aninhaler, spray dispenser (e.g. nasal spray) or pressure pack forcapsules, tables, or other device as described herein.

In another aspect of the invention, kits for treating an individual whosuffers from or is susceptible to respiratory disorders are provided,comprising a container comprising a dosage amount of an multifunctionalβ-agonist compound or composition as disclosed herein, and instructionsfor use.

Kits may also be provided that contain sufficient dosages of themultifunctional β-agonist compound or composition to provide effectivetreatment for an individual for an extended period, such as a week, 2weeks, 3, weeks, 4 weeks, 6 weeks or 8 weeks or more.

The invention is further illustrated by the following nonlimitingexamples.

EXAMPLES Example 1 Synthesis of5-{2-[(1-Yloxyl-2,2,5,5-tetramethyl-pyrrolidin-3-ylmethyl)-amino]-1-S-nitroso-ethyl}-1,1,3,3-tetramethylisoindolin-2-yloxyl,hydrochloride, compound 1

The compound in this example was synthesized as depicted in scheme 4below:

Synthesis of Epoxide I:

The synthesis of epoxide I was performed as described in scheme 3.

2-Benzyl-1,1,3,3,-tetramethylisoindoline

To a solution of methyl Grignard reagent, prepared from methyl iodide(170 g, 1.2 mol) and magnesium turnings 930.4 g, 1.25 mol) in ether (700ml) under nitrogen, was concentrated by slow distillation of solventuntil the internal temperature reached 80° C. The residue was allowed tocool to 60° C., and a solution of N-benzylphthalimide (47.5 g, 0.2 mol)in toluene (600 ml) was added dropwise with stirring at a sufficientrate to maintain this temperature. When the addition was complete,solvent was distilled slowly from the mixture until the temperaturereached 108-110° C. After being maintained at this temperature (reflux)for 4 hours, the mixture was concentrated to approximately 200 ml byfurther solvent distillation. It was then cooled and diluted with lightpetroleum (500 ml). The resulting slurry was filtered through Celite andwashed with light petroleum (3×100 ml), the combined filtrate was keptin an open flask for approx. 2 h and then passed through a short columnof basic alumina (activity 1). The column was further eluted with lightpetroleum until eluent was amine free. Evaporation of the pale yellowsolution gave an oil which rapidly solidified (20 g 37%). Twocrystallizations from methanol or ethanol gave colourless needles of2-benzyl-1,1,3,3,-tetramethylisoindoline. m.p. 63-64° C. NMR (CDCl₃, 400MHz) 1.3, s, 4×CH₃; 4.05, s, ArCH2; 7.04-7.54, m, ArH.

1,1,3,3-Tetramethylisoindoline

A solution of 2-benzyl-1,1,3,3,-tetramethylisoindoline (5.5 g 20 mmol)in glacial acetic acid (20 ml) was hydrogenated at 60 lb/in² over 5%palladium/c for 3 h at room temperature. The suspension was filtered andthe solvent removed at reduced pressure. The residue was dissolved inwater and the solution was made alkaline (PH 9) by the addition of 10%sodium hydroxide and extracted with ether (3×50 ml). The combinedorganic extracts were dried and evaporated to dryness to give the titlecompound (3.5 g 96%), which was recrystallized from methanol/water. m.p.36-38° C. NMR (CDCl₃, 400 MHz), 1.46 s, 4×CH₃; 1.9 s, NH, D₂O exchanged;7.1-7.27 m, ArH.

1,1,3,3-Tetramethylisoindoline-2-yloxyl

A solution of 1,1,3,3-tetramethylisoindoline (3.3 g 18.8 mmol) inmethanol (35 ml) and acetonitrile (2.5 ml) was added sodium hydrogencarbonate (1.26 g, 15 mmol), sodium tungstate dihydrate (0.18 g, 0.55mmol) and finally 30% aqueous hydrogen peroxide (7 ml, 62 mmol). Thesuspension was stirred at room temperature for 32 h to give a brightyellow solution, which was diluted with distilled water and extractedwith light petroleum (3×75 ml). The combined organic layers were washedwith 2N sulfuric acid and followed by brine. Drying and evaporation ofthe solvent gave 1,1,3,3-tetramethylisoindoline-2-yloxyl (3.1 g, 92%) asbright yellow solid, which was recrystallized from light petroleum. m.p.128-129° C. m/e (70 eV) 190 (M, 38%).

1-(2-Yloxyl-1,1,3,3-tetramethyl-isoindoline-5-yl)-ethanone

AlCl₃ (1.6 g, 12 mmol) was added to a solution of1,1,3,3-tetramethylisoindoline-2-yloxyl (1.9 g, 10 mmol) in chloroform(100 ml). To this suspension, acetyl chloride (0.86 g, 11 mmol) wasadded dropwise with stirring. When the addition was complete, thereaction mixture was refluxed for 2 h, cooled to room temperature andcrushed ice was added. The organic phase was separated and washed twicewith 1N NaOH (50 ml) and once with brine. The organic phase was driedand evaporated to dryness. The crude product was chromatographed onsilica gel with hexane:ethylacetate (9:1) to give the pure product1-(2-yloxyl-1,1,3,3-tetramethyl-2,3-dihydro-1H-isoindol-5-yl)-ethanone(2.18 g, 95%). M⁺ 232.

2-Bromo-1-(2-yloxyl-1,1,3,3-tetramethyl-isoindolin5-yl)-ethanone

To a solution of 0.1 mol of1-(2-yloxyl-1,1,3,3-tetramethyl-isoindolin-5-yl)-ethanone and 0.1-0.2 gof dibenzoyl peroxide in 250 ml of CHCl₃ was added dropwise 1 equivalentof Br₂. The reaction mixture was stirred at room temperature until Br₂color had disappeared, nitrogen was bubbled through the solution for15-20 minutes, and the solvent was evaporated in vacuum. The residue wastriturated with ether or recrystallized from the appropriate solvent toyield the corresponding bromoketone.

5-(2-Bromo-1-hydroxy-ethyl)-1,1,3,3-tetramethyl-isoindolin-2-yloxyl

Reduction of2-bromo-1-(2-yloxyl-1,1,3,3-tetramethyl-isoindolin-5-yloxyl)-ethanonewas achieved with borane and borane/oxazaborolidine (R or S) in THF toyield nonchiral and chiral bromohydrin, respectively. The product wasobtained in 90-95% yield after purification by column chromatography(ethylacetate:hexane 1:4).

1,1,3,3-Tetramethyl-5-oxiranyl-isoindoline-2-yloxyl (1)

Treatment of5-(2-Bromo-1-hydroxy-ethyl)-1,1,3,3-tetramethyl-isoindolin-2-yloxyl withsuspension of NaH in THF generated the title epoxide in 85% yield.

1,1,3,3-Tetramethyl-5-thiiranyl-1,3-dihydro-isoindol-2-ol (II)

Epoxide (I) (10 mmol) was added to a solution of sodiumthiocyanate (12mmol) in water (10 ml) and ethanol (7.5 ml), the solution was vigorouslystirred at room temperature for 36 h. After cooling, the precipitatedsodium cyanate was filtered off and the filtrate was extracted withether, washed twice with brine, dried with sodium sulfate and evaporatedto dryness. The residue was chromatographed on silica gel(ethylacetate:hexane, 1:9).

Synthesis of the amine III,3-Aminomethyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl

This compound was synthesized according to the scheme 5 below:

3-Hydroxymethyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl

To an ice cooled suspension of 2,2,5,5-tetramethylpyrrolidine carboxylicacid (18.4 g, 0.1 mol) in dry ether (250 ml) and triethylamine (14 ml,0.1 mol), ethylchloroformate (10.0 g, 0.1 mol) was added with vigorousstirring. The reaction mixture was stirred at 0° C. for 2 h. Ice coldwater (100 ml) was then added and the organic phase was washed with 100ml of ice cold 1N HCL (100 ml), water (100 ml), 5% Na₂CO₃ solution (100ml) and water. The organic phase was dried and evaporated to dryness togive (1 g, 70%) of the mixed anhydride as a red oily product, which wasreduced in an ethanolic suspension of NaBH₄ (3.12 g, 1.2 eq) at roomtemperature for 1 h. Ethanol was evaporated and the residue waspartitioned between water and ether. The organic phase is separated andwashed with distilled water and brine, dried and evaporated to drynessto give 3-Hydroxymethyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl (9.7 g,56%), which can be recrystallized with ether-hexane.

3-Tosyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl

To a stirred pyridine solution of3-Hydroxymethyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl (1.7 g, 10mmol), that was cooled to 0° C., para-toluenesulfonyl chloride (1.9 g,10 mmol) was added in three portions and the reaction mixture is stirredfor further 3 h at room temperature. Pyridine was evaporated and theresidue was dissolved in chloroform, washed twice with 1N HCl, 5% sodiumcarbonate solution and water. The organic phase was dried with sodiumsulfate and evaporated to dryness to give a yellow crystals of3-Tosyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl (3 g 91%) which can berecrystallized from chloroform/hexane.

3-Azidomethyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl

Sodium azide (0.975 g, 15 mmol) and3-Tosyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl (3.27 g, 10 mmol) inacetonitrile (100 ml), were reflux for three hours, and cooled to roomtemperature. The reaction was filtered and the acetonitrile wasevaporated to dryness. The residue was dissolved in chloroform andwashed twice with distilled water, dried and evaporated to dryness toafford the title compound (1.8 g, 91%) that can be recrystallized fromchloroform hexane.

3-Aminomethyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl

To 3-Azidomethyl-2,2,5,5-tetramethyl-pyrrolidin-1-yloxyl (1.97 g, (10mmol) in 70% ethanol, triphenyl phosphine (1.5 eq) was added and thesolution was stirred at room temperature for 4 h. after the reaction wascompleted, it was extracted with ether and the ether fraction was washedtwice with distilled water and brine. The ether was dried with sodiumsulfate and evaporated to dryness to give a red solid residue which wascrystallized from chloroform hexane (1.5 g, 87%).

5-{2-[(1-Yloxyl-2,2,5,5-tetramethyl-pyrrolidin-3-ylmethyl)-amino]-1-S-nitroso-ethyl}-1,1,3,3-tetramethylisoindolin-2-yloxyl,hydrochloride (compound 1)

A mixture of compound III (10 mmol) and 1 ml ofN,O-bis-(trimethylsilyl)-acetamide in 50 ml of DMSO was stirred undernitrogen for 30 minutes at room temperature. A solution of compound IIin 50 ml of DMSO was added and the solution was heated at 80° C. for 48h. the mixture was cooled and the solvent was vacuum distilled (0.1 mm).The residue was purified by column chromatography on neutral aluminaeluting with ethylacetate:hexane (1:3). This compound IV is dissolved in10 ml of 8N HCl, stirred and cooled in an ice bath. Three molequivalents of sodium nitrite is dissolved in distilled water and addeddropwise to the reaction mixture. After 20 minutes, when most of thesodium nitrite was added, the title S-nitroso compound V/compound 1separated as pale orange powder. Stirring is continued for 30 minutes inthe cold bath and the precipitated product is filtered, washed twicewith cold diluted HCl, dried in the desecrator and stored in airtightcontainers under nitrogen at −20° C. until use. MS and spectroscopicdata of this compound V/compound 1 were found as expected for S-nitrosothiols.

Example 2 Synthesis of5-(2-tert-Butylamino-1-S-nitroso-ethyl)-1,1,3,3-tetramethyl-isoindolin-2-yloxyl,compound 2

This compound was prepared as described in the following scheme:

A mixture of tert-butylamine (10 mmol) and 1 ml ofN,O-bis-(trimethylsilyl)-acetamide in 50 ml of DMSO was stirred undernitrogen for 30 minutes at room temperature. A solution of the thiirane(compound II) in 50 ml of DMSO was added and the solution was heated at80° C. for 48 h. the mixture was cooled and the solvent was vacuumdistilled (0.1 mm). The thiol-containing residue 2a was purified bycolumn chromatography on neutral alumina eluting withethylacetate:hexane (1:4). This compound 2a is dissolved in 10 ml of 8NHCl, stirred and cooled in an ice bath. Three mol equivalents of sodiumnitrite is dissolved in distilled water and added dropwise to thereaction mixture. After 20 minutes, when most of the sodium nitrite wasadded, the title S-nitroso compound 2 separated as a pale orange powder.Stirring is continued for 30 minutes in the cold bath and theprecipitated product is filtered, washed twice with cold diluted HCl,dried in the dessicator and stored in airtight containers under nitrogenat −20° C. until use. MS and spectroscopic data of this compound 2 werefound as expected for S-nitroso thiols.

Example 3 Synthesis of5-[1-Hydroxy-2-(2-S-nitroso-1-methyl-2,2-diphenyl-ethylamino)-ethyl]-1,1,3,3-tetramethyl-isoindoline-2-Yloxyl,compound 3

This compound was synthesized as illustrated in the following scheme:

The amine utilized for this synthesis was synthesized as described inthe following scheme:

(S)-(−)-2-Amino-1,1-diphenyl-propan-1-ol was converted to(S)-(−)-2-amino-1,1-diphenyl-propane-1-thiol by refluxing inconcentrated aqueous HBr with 4 mol equivalents of thiourea for 36 h.After cooling, the reaction mixture was neutralized with excess KOH andrefluxed for 8 h under inert conditions (nitrogen). The product wasextracted with ether and purified by column chromatography on neutralalumina (ethylacetate:hexane 1:3).

5-[1-Hydroxy-2-(2-S-nitroso-1-methyl-2,2-diphenyl-ethylamino)-ethyl]-1,1,3,3-tetramethyl-isoindoline-2-Yloxyl(compound 3)

A mixture of the amine thiol (10 mmol) and 1 ml ofN,O-bis-(trimethylsilyl)-acetamide in 50 ml of DMSO was stirred undernitrogen for 30 minutes at room temperature. A solution of the epoxide(compound I) in 50 ml of DMSO was added and the solution was heated at80° C. for 48 h. the mixture was cooled and the solvent was vacuumdistilled (0.1 mm). The residue was purified by column chromatography onneutral alumina with ethylacetate:hexane (1:3). This compound isdissolved in 10 ml of 8N HCl, stirred and cooled on an ice bath. Threemol equivalents of sodium nitrite is dissolved in distilled water andadded dropwise to the reaction mixture. After 20 minutes, when most ofthe sodium nitrite was added, the title S-nitroso compound 3 separatedas pale orange powder. Stirring is continued for additional 30 minutesin the cold and the precipitated product is filtered, washed twice withcold diluted HCl, dried in the dessicator and stored in airtightcontainers under nitrogen at −20° C. until use. MS and spectroscopicdata of this compound were found as expected for S-nitrosothiols.

Example 4 Synthesis of5-[2-(4-amino-2,2,6,6-tetramethyl-piperidin-1-yloxyl)-1-S-nitrosothiol-ethyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl(compound 4)

This compound was synthesized as shown in the following scheme:

The amine (4-amino-TEMPO) was synthesized according to the followingscheme (NaTg is Na₂WO₄):

4-Acetylamino-2,2,6,6-Tetramethyl-piperidin

Acetic anhydride (70 g, 0.686 mol) was added dropwise to an ice cooledsolution of 4-Amino-2,2,6,6-Tetramethyl-piperidin (34.6 g. 0.221 mol) inether (100 ml). After the addition was complete (about 1 h), thereaction mixture was stirred for 30 minutes at room temperature. Theprecipitated white product, 4-acetylamino-2,2,6,6-Tetramethyl-piperidin(55.6 g, 98%) was filtered off and washed with ether and air dried. m.p.175° C. subl.

4-Acetylamino-2,2,6,6-tetramethyl-piperidin-1-yloxyl

55.6 g of 4-acetylamino-2,2,6,6-Tetramethyl-piperidin was dissolved in400 ml of water and basified with 50 g of potassium carbonate. To thissolution was added 80 ml of 30% hydrogen peroxide, 4 g of sodiumtungstenate (NaTg) and 4 g of EDTA. The mixture was stirred at roomtemperature for 72 h and the red precipitate filtered off and washedwith distilled water to give the title compound (36.8 g). The filtratewas further extracted with dichloromethane to give more 7.1 of theproduct (overall yield 97%). m.p. 145-147° C.

4-Amino-2,2,6,6-tetramethyl-piperidin-1-yloxyl

11 g of 4-acetylamino-2,2,6,6-Tetramethyl-piperidin-1-yloxyl washydrolyzed by refluxing in 25 ml of 15% KOH for 12 h. The reactionmixture was saturated with potassium carbonate and extracted with ether(2×50 ml). The ether was dried with sodium sulfate and the crude productwas vacuum distilled (97-98/4 mm Hg) to give the pure4-amino-2,2,6,6-Tetramethyl-piperidin-1-yloxyl (8 g, 73%) as a deep redcrystals. m.p. 34-35° C.

5-[2-(4-amino-2,2,6,6-tetramethyl-piperidin-1-yloxyl)-1-nitrosothiol-ethyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl(compound 4)

A mixture of 4-amino-TEMPO (10 mmol) and 1 ml ofN,O-bis-(trimethylsilyl)-acetamide in 50 ml of DMSO was stirred undernitrogen for 30 minutes at room temperature. A solution of the thiirane(compound II) in 50 ml of DMSO was added and the solution was heated at80° C. for 48 h. the mixture was cooled and the solvent was vacuumdistilled (0.1 mm). The residue was purified by column chromatography onneutral alumina eluting with ethylacetate:hexane (1:3). This compound isdissolved in 10 ml of 8N HCl, stirred and cooled in an ice bath. Threemol equivalents of sodium nitrite is dissolved in distilled water andadded dropwise to the reaction mixture. After 20 minutes, when most ofthe sodium nitrite was added, the title S-nitroso compound 4 separatedas an orange powder. Stirring is continued for 30 minutes in the coldbath and the precipitated product is filtered, washed twice with colddiluted HCl, dried in the dessicator and stored in airtight containersunder nitrogen at −20° C. until use. MS and spectroscopic data of thiscompound were found as expected for S-nitroso thiols.

Example 5 Synthesis of5-[1-Hydroxy-2-(2-S-nitroso-1,2-diphenyl-ethylamino)-ethyl]-1,1,3,3-tetramethyl-1,3-dihydroisoindolin-2-yloxyl(compound 5)

This compound was synthesized as described in the following scheme:

The amine utilized for this synthesis was synthesized as described inthe following scheme:

(1R,2S)-(−)-2-Amino-1,2-diphenyl-ethanethiol

The starting (1R,2S)-(−)-2-amino-1,2-diphenylethanol was first protectedwith the Boc group using di-tert-butyldicarbonate in dioxane, followedby mesylating the hydroxyl group with methanesulfonyl chloride indichloromethane/triethylamine solution. The mesylate was treated withNaBr in acetone to yield the bromide, which is then treated withthiourea to produce the mercapto compound with the protected amine. Thislater compound was finally treated with trichloroacetic acid to liberatethe amino group and give the title compound.

5-[1-Hydroxy-2-(2-S-nitroso-1,2-diphenyl-ethylamino)-ethyl]-1,1,3,3-tetramethyl-1,3-dihydro-isoindolin-2-yloxyl(compound 5)

A mixture of the amine (10 mmol) and 1 ml ofN,O-bis-(trimethylsilyl)-acetamide in 50 ml of DMSO was stirred undernitrogen for 30 minutes at room temperature. A solution of the epoxide(compound I) in 50 ml of DMSO was added and the solution heated at 80°C. for 48 h. After cooling, the solvent was vacuum distilled (0.1 mm),and the residue purified by column chromatography on neutral aluminawith ethylacetate:hexane (1:3). This compound is dissolved in 10 ml of8N HCl, stirred and cooled in an ice bath. Three mol equivalents ofsodium nitrite is dissolved in distilled water and added dropwise to thereaction mixture. After 20 minutes, when most of the sodium nitrite wasadded, the title S-nitroso compound 5 separated as pale orange powder.Stirring is continued for 30 minutes in the cold bath and theprecipitated product is filtered, washed twice with cold diluted HCl,dried in the dessicator and stored in airtight containers under nitrogenat 20° C. until use. MS and spectroscopic data of this compound werefound as expected for S-nitrosothiols.

Example 6 Synthesis ofN-(5-{2-[(4,4-Dimethyl-[1,2]dithiolan-3-ylmethyl)-amino]-1-nitrooxy-ethyl}-2-hydroxy-phenyl)-formamide,hydrochloride, compound 6

This compound was prepared as illustrated in the following scheme.

The amine used for the synthesis of this compound was synthesized asdescribed in the following scheme:

(+)-4,4-Dimethyl-[1,2]dithiolan-3-yl)-methylamine

(+)-Pantholactone (or DL) was reduced with LiAlH in ether to give3,3-dimethyl-butane-1,2,4-triol in 60% yield, which was protected as a 6membered acetonide by treatment with benzaldehyde in benzene undertrifluoroacetic acid catalysis in 90% yield. The remaining free hydroxylgroup was converted to the amine by the same method used for thepreparation of the amine in example 6, which was further protected withthe Boc group. Deprotection of the hydroxyl groups by catalytichydrogenation followed by dimesylation of the obtained 2,4-diol gave thedimesylate, which was converted to the disulfide using sulfur and sodiumsulfide in DMF under standard dithiolation conditions. Finally, theamine was deprotected with trichloroacetic acid in dichloromethane toafford the title amino compound 6.

The amine was treated with the corresponding epoxide, followed bygeneration of the hydrochloride salt and nitration in the same manner asdescribed for example 7 to yield the hydrochloride salt of the titlecompoundN-(5-{2-[(4,4-dimethyl-[1,2]dithiolan-3-ylmethyl)-amino]-1-nitrooxy-ethyl}-2-hydroxy-phenyl)-formamide.

Example 7 Synthesis of[2-(2,2-Dimethyl-4H-benzo[1,3]dioxin-6-yl)-2-nitrooxy-ethyl]-(5-[1,2]dithiolan-3-yl-pentyl)-aminehydrochloride, 7

This compound was synthesized as illustrated in the following scheme:

The starting materials for the synthesis of this compound weresynthesized as following:

I—2,2-Dimethyl-6-oxiranyl-4H-benzo[1,3]dioxine

First the hydroxyl groups of Salicyl alcohol were protected with acetonein refluxing benzene with the catalysis of para-toluenesulfonic acid togive 2,2-Dimethyl-4H-benzo[1,3]dioxine in quantitive yield.Friedel-craft acylation of this compound with AlCl₃ and acetyl chloridein refluxing chloroform afforded1-(2,2-dimethyl-4H-benzo[1,3]dioxin-6-yl)-ethanone in good yield. Thiswas further brominated in chloroform with the catalysis of benzoylperoxide to give2-bromo-1-(2,2-dimethyl-4H-benzo[1,3]dioxin-6-yl)-ethanone, which wasreduced to the chiral or nonchiral bromohydrine with borane andborane/oxazaborolidine, respectively. The title epoxide was generated bythe addition of the above bromohydrine to a suspension of NaH in THF, asdescribed previously.

II—5-[1,2]Dithiolan-3-yl-pentylamine (lipoyl amine)

Lipoyl alcohol was obtained as a yellow oil in 95% yield by thereduction of lipoic acid with borane-THF complex in dry THF, andpurification by column chromatography. The alcohol was converted to thetosylate with paratoluenesulfonyl chloride under standard conditions.The azide compound was generated by the reaction of the tosylate withNaN₃ in acetonitrile. Finally, the lipoyl amine was obtained by reactionof the lipoyl azide with triphenylphosphine in 70% aqueous ethanol. Theoverall yield of lipoyl amine (from lipoic acid) is in the range of75-80%.

[2-(2,2-Dimethyl-4H-benzo[1,3]dioxin-6-yl)-2-nitrooxy-ethyl]-(5-[1,2]dithiolan-3-yl-pentyl)-amine(compound 7)

A mixture of lipoyl amine (10 mmol) and 1 ml ofN,O-bis-(trimethylsilyl)-acetamide in 50 ml of DMSO was stirred undernitrogen for 30 minutes at room temperature. A solution of the epoxidein 50 ml of DMSO was added and the solution was heated at 80° C. for 48h. After cooling, the solvent was vacuum distilled (0.1 mm) and theresidue was purified by column chromatography on neutral alumina withethylacetate:hexane (1:4). This compound is converted to thehydrochloride salt by bubbling of gaseous HCl to its methanolic solutionand precipitating with ether. The title nitro compound 7 was obtained bybubbling nitrogen dioxide gas in THF solution of the hydrochloride saltwith stirring. The product was stored in airtight containers undernitrogen at −20° C. until use.

Example 8 Synthesis ofN-{5-[2-(5-[1,2]Dithiolan-3-yl-pentylamino)-1-nitrooxy-ethyl]-2-hydroxy-phenyl}-formamidehydrochloride, compound 8

This compound was synthesized according to the following scheme:

The epoxide (2-(4-Benzyloxy-3-nitro-phenyl)-oxirane) utilized for thesynthesis of this compound was synthesized as described in the followingscheme:

A mixture of lipoyl amine and the epoxide were heated at 120° C. untilall epoxide has disappeared (monitored by TLC), followed by selectivereduction of the nitro group in formic acid afforded the formamide thatwas purified by chromatography and hydrogenated to deprotect thehydroxyl group. This compound was converted to the hydrochloride salt bytreatment with gaseous HCl in methanol and precipitated with ether,Treatment of the obtained salt in THF with nitrogen dioxide afforded thetitle compound 8.

Example 9 Synthesis of5-[1-Hydroxy-2-(4-S-nitroso-4-methyl-cyclohexylamino)-1,1,3,3-tetramethyl-1,3-dihydro-isoindol-2-yloxy,free radical, compound 9

This compound was synthesized by reacting epoxide I in Example 1 withthe corresponding amine followed by hydrochloride salt formation andS-nitrosylation by either sodium nitrite or ethyl nitrite. The amine(4-mercapto-4-methyl cyclohexylamine) was synthesized from thecommercially available 4-hydroxy cyclohexylamine by standard methodsinvolving amine protection (Boc), oxidation, thiolation and reactingwith methyl magnesium iodide. The resulting N-protected4-mercapto-4-methyl cyclohexylamine was deprotected and reacted withepoxide I as described above.

Example 10 Synthesis of5-{1-Hydroxy-2-[1-S-nitroso-1-methyl-ethyl)-cyclohexylamino]-ethyl}-1,1,3,3-tetramethyl-1,3-dihydro-isoindol-2-yloxy,free radical, compound 10

This compound was synthesized by reacting epoxide I in Example 1 withthe corresponding amine followed by hydrochloride salt formation andS-nitrosylation by either sodium nitrite or ethyl nitrite, as describedabove. The amine (8-mercapto menthylamine) was synthesized from thecommercially available 8-mercapto menthone by standard methods involvingS-protection and reductive amination. The resulting S-protectedmenthylamine was deprotected and reacted with epoxide I as describedabove.

Example 11 Biological Activity

The potency and efficacy of the multifunctional β-agonist compounds areevaluated using a model of biological response for asthma as describedbelow, where increased relaxation is an in vitro indication of increasedefficacy, and is a predictive model for in vivo efficacy. The assays asdescribed here are also described in, for example, U.S. Pat. No.4,992,474 and Jansen et al. (1992) J. Pharmacol. Exptl. TherapeuticsI261(1):154-160.

Tissue Preparation

Male Hartley guinea pigs (500-600 g) were anesthetized byintraperitoneal injection of ketamine and xylazine (50 and 10 mg/kg,respectively). The heart and lungs were excised en bloc and tracheaswere removed and placed in Krebs-Henseleit buffer composed of (mM): NaCl118, KCl 5.4, NaH₂PO₄ 1.01, NaHCO₃ 25, MgSO₄ 0.69, CaCl₂ 2 2.32, glucose11.1, pH 7.4. Tracheas were then dissected free from surrounding fat andconnective tissue and cut into 1-2-mm thick rings. The tracheal ringswere then placed in buffer and continuously gassed with 95% O₂ and 5%CO₂.

Relaxation Studies

Tracheal rings were suspended on stainless steel hooks in 10 ml ofoxygenated (95% O₂, 5% CO₂) Krebs-Henseleit buffer at 37° C. andconnected to transducer (Experimetria Model) for recording changes inisometric force. The tracheal rings were equilibrated for 60 min under aload of 1 g and then primed twice by exposure to 100 μM methacholine.Tissues were contacted with methacholine, histamine, or leukotriene D₄at concentrations determined to approximately generate 50% of maximaltone, after which cumulative relaxation-response curves wereconstructed. To construct these curves, the initial contraction wasassigned a value of 100% and the bath concentration of the testedcompound required to achieve 50% relaxation (i.e., IC₅₀) determined bylinear interpolation.

FIG. 3 shows the results with salbutamol (open squares); compound 2(filled circles); and its non-nitrosylated precursor, compound 2a (opentriangles). As shown in FIG. 3, compound 2 is a more effective relaxantcompound than either salbutamol or the non-nitrosolated precursor ofcompound 2, compound 2a, which incorporates the ROS scavenger andβ-agonist moiety of salbutamol, but not the NO donor moiety. Thus,compound 2 is much more effective at a lower concentration than eithercompound 2a or salbutamol.

Cyclic Nucleotide Assays

Tracheal rings in Krebs-Heneseleit solution were exposed to a 5-100 μMof the test compound for 30-90 seconds. Reaction was terminated by theaddition of ice-cold 10% trichloroacetic acid and rapidly frozen inethanol-saturated dry ice. In the methylene blue experiment, rings werepre-exposed methylene blue, a guanylyl cyclase inhibitor, for 30 min.Tissues were then individually pulverized with a glass homogenizer andcentrifuged at 8000×g for 5 min. The clear supernatant was extractedwith water-saturated ether and assayed for cGMP using commerciallyavailable radioimmunoassay kits as described by the manufacturer(Amersham Biosciences, Piscataway, N.J.).

As indicated by FIG. 4, compound 2 elevates the production of cGMP,indicative of NO-guanylyl cyclase pathway activity. Compound 2a, thenon-nitrosolated precursor of compound 2, does not stimulate theproduction of cGMP compared to the control compound or salbutamol.Incubation with methylene blue, a guanylyl cyclase inhibitor, reducesthe efficacy of compound 2, as would be expected for a mechanism ofaction which includes NO-guanylyl cyclase pathway activity. From theseresults it can be seen that the added activity of compound 2 compared tocompound 2a is due to the NO-donating properties of compound 2.

All publications, patents, and patent applications referred to hereinare hereby incorporated herein by reference in their entirety.

1-38. (canceled)
 39. A method of treating or preventing a chronicobstructive airway disease in a mammal in need thereof comprisingadministering to said mammal an effective amount of a multifunctionalβ-agonist compound being ROS scavenger and NO donor of Formula 1:

or a salt thereof or a solvate thereof or an optical isomer thereof,wherein R¹ is selected from the group consisting of —OH, —ONO, —ONO₂,—SNO, and —NONOate; R² is ROS scavenger group or a NO donor groupconnected to the —NH group via a linker made of C₅-C₈ cyclic alkyl, orstraight or branched C₁-C₁₅ alkyl in which one carbon atom is optionallyreplaced by oxygen or nitrogen, wherein said ROS scavenger group isselected from a nitroxide free radical, alkenyl, sulfhydryl or dithiolmoiety in oxidized or reduced form, and aryl, and wherein said NO donorgroup is selected from —ONO, —ONO₂, —SNO, and —NONOate or R² is C₅-C₈cyclic alkyl, or straight or branched C₁-C₁₅ alkyl; R³ and R⁴ areindependently selected from the group consisting of —OH, —CH₂OH, —NH₂,—NHCHO, or R³ and R⁴ together form a substituted 5 to 7-memberedsaturated heterocycle having 1 or 2 heteroatoms independently selectedfrom nitrogen, and oxygen, and sulfur, or R³ and R⁴ together form aminoor hydroxy protecting groups selected from N-formyl, acetal, and ketal;R⁵ is selected from the group consisting of —H, —OH, —CH₂OH, —NH₂,—NHCHO, straight or branched chain C₁-C₁₅ alkyl, and straight orbranched chain C₁-C₁₅ alkoxy; whereas said ROS scavenger moieties areoptionally substituted with one or more C₁-C₁₅ alkyl groups, C₁-C₁₅alkoxy groups, phenyl, —NH₂, —NHCHO, —OH, —CH₂OH, and groups capable ofdonating NO in a charged or neutral form; and whereas any of said alkylgroups is optionally substituted with one or more functional groupsselected from hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio,cyano, alkylthio, aryl, carboxyl, carbalkoyl, alkenyl, nitro, amino,alkoxyl, amido; wherein at least one of R¹, R², R³ and R⁴ comprises atleast one ROS scavenger selected from the group of moieties consistingof a nitroxide free radical, alkenyl, sulfhydryl or dithiol in oxidizedor reduced form, and aryl; and wherein at least one of R¹, R², R³ and R⁴comprises at least one NO donor selected from —ONO, —ONO₂, and —SNO. 40.A method according to claim 39, wherein said saturated heterocycle isselected from the group consisting of pyrrolidine, oxazolidine,thiazolidine, tetrahydro-1,3-oxazine, 1,3-dioxane, piperidine,3-thiapiperidine, and 1,3-thiazine.
 41. A method according to claim 39,wherein said saturated heterocycle comprises a substituted nitroxidefree radical.
 42. A method according to claim 39, wherein the nitroxidefree radical is a heterocyclyl moiety having the nitrogen atom within a5-, 6- or 7-membered ring which optionally contains another heteroatomselected from oxygen and sulfur at position beta to the nitrogen, andwhich is substituted with methyl or ethyl at positions alpha to thenitrogen.
 43. A method according to claim 42, wherein said heterocyclylmoiety is linked to the β-agonist moiety via sharing of 1 to 2 atoms, orvia a linker.
 44. A method according to claim 39, wherein said ROSscavenger group is selected from the group consisting of the followingmoieties:

wherein X is selected from carbon, oxygen, and sulfur, and n is aninteger from 1 to
 15. 45. A method according to claim 39, wherein R² isselected from the following structures:

wherein m is 1-6 and R⁸ and R⁹ are independently C₁-C₃ alkyl or —H. 46.A method according to claim 39, wherein said multifunctional β-agonistcompound has the formula:

or its salt; wherein R¹ is selected from the group consisting of —OH,—ONO, —ONO₂, and —SNO; R⁵ is hydrogen; and R² is a moiety selected froma nitroxide free radical having the nitrogen atom within a 5-, 6- or7-membered saturated ring and which is substituted by up to four methylgroups at positions alpha to the nitrogen, sulfhydryl or dithiol moietyin oxidized or reduced form, —ONO, —ONO₂, and —SNO, wherein said moietyis connected to the —NH group directly or via a linker made of C₁-C₆alkyl, and which linker is optionally substituted by one or more phenylgroups.
 47. A method according to claim 39, wherein said multifunctionalβ-agonist compound has one of the following structures:


48. A method according to claim 39, wherein said chronic obstructiveairway disease is selected from the group consisting of asthma andchronic obstructive pulmonary disease.
 49. A method according to claim48, comprising symptoms selected from the group consisting of emphysema,chronic bronchitis, recurrent obstruction to air flow within the lung,increased resistance to air flow, narrowing or restriction of an airway,inflammation, bronchial hyperreactivity, airway hyperresponsiveness,mucosal edema, mucus plugging and hypersecretion, and reduced expansionof respiratory parenchyma.
 50. A method according to claim 48, whereinsaid asthma is selected from the group consisting of atopic, extrinsic,and intrinsic.
 51. A method according to claim 39, wherein saidadministration is selected from the group consisting of systemicadministration and topical administration.
 52. A method according toclaim 39, wherein said β-agonist compound is administered by a routeselected from the group consisting of oral, parenteral, intramuscular,intraperitoneal, intravenous, ICV, intracisternal injection or infusion,subcutaneous injection, implant, buccal, inhalation spray, nasal,vaginal, rectal, and sublingual route.
 53. A method of claim 39, whereinsaid mammal is human.
 54. A method according to claim 39, wherein saidβ-agonist compound, or a salt thereof or a solvate thereof or an opticalisomer thereof is administered in a pharmaceutical compositioncomprising carriers, adjuvants, and excipients.
 55. A method accordingto claim 54, said composition further comprising an active agentselected from the group consisting of mucolytic, bronchodilator, musclerelaxant, decongestant, respiratory stimulant, vasodilator, β-agonist,anti allergic, antiasthmatics, analgesic, anti-inflammatory, antibiotic,antifungal, antiprotozoal, and antiviral agent.
 56. A method accordingto claim 52, wherein said administration is via an inhalation device.57. An inhalation device for administering a multifunctional β-agonistcompound according to the method of claim 39, said compound being ROSscavenger and NO donor of Formula 1:

or its salt, wherein R¹ is selected from the group consisting of —OH,—ONO, —ONO₂, and —SNO; R² is ROS scavenger group or a NO donor groupconnected to the —NH group via a linker made of C₅-C₈ cyclic alkyl orstraight or branched C₁-C₁₅ alkyl in which one carbon atom is optionallyreplaced by oxygen or nitrogen, wherein said ROS scavenger group isselected from a nitroxide free radical, alkenyl, sulfhydryl or dithiolmoiety in oxidized or reduced form, and aryl, and wherein said NO donorgroup is selected from —ONO, —ONO₂, and —SNO, or R² is C₅-C₈ cyclicalkyl, or straight or branched C₁-C₁₅ alkyl; R³ and R⁴ are independentlyselected from the group consisting of —OH, —CH₂OH, —NH₂, —NHCHO, or R³and R⁴ together form a substituted 5 to 7-membered saturated heterocyclehaving 1 or 2 heteroatoms independently selected from nitrogen, andoxygen, and sulfur, or R³ and R⁴ together form amino or hydroxyprotecting groups selected from N-formyl, acetal, and ketal; R⁵ isselected from the group consisting of —H, —OH, —CH₂OH, —NH₂, —NHCHO,straight or branched chain C₁-C₁₅ alkyl, and straight or branched chainC₁-C₁₅ alkoxy; whereas said ROS scavenger moieties are optionallysubstituted with one or more C₁-C₁₅ alkyl groups, C₁-C₁₅ alkoxy groups,phenyl, —NH₂, —NHCHO, —OH, —CH₂OH, and groups capable of donating NO ina charged or neutral form; and whereas any of said alkyl groups isoptionally substituted with one or more functional groups selected fromhydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano,alkylthio, aryl, carboxyl, carbalkoyl, alkenyl, nitro, amino, alkoxyl,amido; wherein at least one of R¹, R², R³ and R⁴ comprises at least oneROS scavenger selected from the group of moieties consisting of anitroxide free radical, alkenyl, sulfhydryl or dithiol in oxidized orreduced form, and aryl; and wherein at least one of R¹, R², R³ and R⁴comprises at least one NO donor selected from —ONO, —ONO₂, and —SNO;which device comprises an element selected from the group consisting ofmetered dose inhaler, liquid nebulizer, dry powder inhaler, sprayer, andthermal vaporizer.
 58. A kit comprising an inhalation device accordingto claim 57, wherein said multifunctional β-agonist is in the form offine power or solution or suspension, wherein said powder or solution orsuspension optionally contains other components selected from bulkingagent, buffer, carrier, excipient, additive, antioxidant, stabilizer,surfactant, odorant, and a second pharmaceutically active agent.
 59. Amethod of treating or preventing a respiratory disorder in a mammal inneed thereof comprising administering to said mammal an effective amountof a multifunctional β-agonist compound as described in claim 39, or asalt thereof or a solvate thereof or an optical isomer thereof.
 60. Amethod according to claim 59, wherein said disorder is selected from thegroup consisting of asthma, chronic bronchitis, bronchiectasis,emphysema, chronic obstructive pulmonary disease, chronic obstructiveairway disease, acute respiratory distress syndrome (ARDS) or severeacute respiratory syndrome (SARS) in child or adult, pneumonia,pneumonitis, and restrictive diseases of the lungs.